synthesis of novel bioactive doxycycline derivatives
TRANSCRIPT
Synthesis of Novel Bioactive Doxycycline
Derivatives
Der Naturwissenschaftlichen Fakultät
der Friedrich – Alexander – Universität Erlangen – Nürnberg
zur
Erlangung des Doktorgrades
vorgelegt von
Igor Usai aus Cagliari (Italien)
Als Dissertation genehmigt von der Naturwissen- schaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung : 29.07.2008
Vorsitzender der Promotionskommission : Prof. Dr. E. Bänsch
Erstberichterstatter : Prof. Dr. P. Gmeiner
Zweitberichterstatter : Prof. Dr. R. Troschütz
The present work has been conducted at the Chair of Medicinal Chemistry of the Friedrich-Alexander-
University Erlangen-Nürnberg under the supervision of
Professor Dr. Peter Gmeiner.
I would like to thank him for his mentorship and guidance, for the helpful discussions and excellent
suggestions that made this work possible.
I thank him for the possibility he gave me to pursue this experience, which made me grow not only
professionally but most importantly as a person.
Thanks go equally to Professor Dr. Reinhard Troschütz for writing the second certificate and to
Professor Dr. Svetlana Tsogoeva for the examination in organic chemistry.
I would like to extend my gratitude to Prof. Dr. Wolfgang Hillen, Dr. Christian Berens, Dr. Oliver
Scholz, Janko Daam and Cornelius Wimmer for the profitable collaboration in the project SFB473 and
for helpful discussions; Prof. Dr. Michael Petz and Ulrike Andree for the collaboration and the tests
within the SPR project.
A special thank goes to Dr. Stefan Löber and to Dr. Jürgen Einsiedel. They were a fundamental
reference point for me during these years. They always listened to my complaints and frustrations,
motivated me and shared with me their ideas and experience. Moreover, I would like to give extra
acknowledgements to Dr. Löber for the critical review of the beta version of my thesis.
Dr. Reiner Waibel was of particular help for unravelling the consequences of the electromagnetic
dance of my compounds; I thank him for his patience and competence.
Thanks to Dr. Wolfgang Utz for the good team-work in mentoring the 1. semester laboratory students
and for his help with computer problems. For his competence and help in computer matters I also
thank Steffen Härterich.
I thank Dr. Harald Hübner for his kindness and for the nice talks we had together.
Thanks to M. Bögelein and M. Schaper for the help in solving the everyday bureaucratic problems,
especially during the first months as “stranger in a strange land”, and for enjoyable chats.
Thanks to I. Torres-Berger, A. Seitz, R. Höfner-Stich, S. Burkhardt, C. Fischer, A. Zillich-Baltasar, R.
Köppl and K. Thomas for their technical support.
I would like to say more than just thanks to Dr. Silke Dollinger, Dr. Luelak Lomlim, Dr. Marika Skultety,
Dr. Jan Elsner, Matthias Horner, Stefan Bollinger, Dr. Christian Kormann and Dr. Miriam Dörfler. Their
friendship helped me overcome these years, and somehow made me feel the home distance a little
shorter. Matthias and Stefan, I thank you for the strength you gave me during the final months of my
thesis, and for the translation of the summary into german.
I would like also to thank all other colleagues that shared a part of their professional life with me.
Thanks to my good old friends Ste, Vale, Corra, Ricky, Robi, Addy, Simone for supporting me during
these years despite the dividing distance.
Thanks to my grandparents, my parents and my brother that supported me with their love and
encouragement. This work is dedicated to You, because you taught me to trust myself and to face
life’s toughness with a smile.
Anna, no words can express my love for you. Thanks for being such a wonderful creature.
Finally, I would like to say farewell to my grandparents Mario Usai and Linda De Poli, who passed
away during my stay in Germany. Thanks for the love you gave me and for your presence in my
childhood. I will always keep Your memory alive.
Erlangen, 23 June 2008.
Teile der vorliegenden Arbeit wurden veröffentlicht :
C. Berens, S. Lochner, S. Löber, I. Usai, A. Schmidt, L. Drueppel, W. Hillen, P. Gmeiner. Subtype Selective Tetracycline Agonists and their Application for a Two-Stage Regulatory System. ChemBioChem 2006 7, 1320-1324.
Konferenzpräsentationen:
Usai, I. ; Einsiedel, J. and Gmeiner, P. “Synthesis of New Doxycycline Derivatives via Click Chemistry” DPhG Jaherestagung, Erlangen 10-13 October 2007 Usai, I. ; Einsiedel, J. and Gmeiner, P. “Synthesis of New Doxycycline Derivatives via Click Chemistry” Frontiers in Medicinal Chemistry (GDCh Jahrestagung), Regensburg March 2-5, 2008 “Synthesis of New Doxycycline Derivatives via Click Chemistry” SFB 473 Berichtskolloquium, Bamberg 7.4.2008
“Always look on the bright side of life”
E. Idle
Table of Contents
1. Introduction 1
2. Background and aims 5
3. Synthesis of new 4-dedimethylamino doxycycline derivatives 14
3.1 4-Dedimethylamino Doxycycline Derivatives 16
3.1.1 Synthesis of 4-Dedimethylamino Doxycycline 16
3.1.2 Substitutions in Position 9 17
3.1.3 Further modifications of 4-Dedimethylamino Doxycycline 19
3.1.4 Further modifications of 9-iodo-4-Dedimethylamino Doxycycline 26
3.2 Biological Investigations 35
4. Synthesis of doxycycline derivatives for SPR investigations 40
4.1 Chemistry 41
4.1.1 Synthesis of amino bearing doxycycline derivatives 41
4.2 Biological Investigations 49
5. Development of a click chemistry strategy for the functionalization and bioconjugation of doxycycline 54
5.1 Chemistry 54
5.1.1 Alkyne / azide derivatives and reaction optimization 54
5.1.2 Functional groups tolerance studies 60
5.1.3 Amino acid conjugates 62
5.1.4 Peptide conjugates 69
5.2 Biological Investigations 78
6. Summary 81
7. Zusammenfassung 93
8. Experimental part 105
9. Abbreviations and acronyms 196
10. References 197
Introduction
1
1. Introduction Tetracyclines are a group of broad-spectrum antibiotics naturally produced by diverse
Streptomyces and Dactylosporangium species, gram-positive bacteria of the family of
Actinomycetes. Tetracycline history goes back to 1947, when Benjamin M. Duggar,
working at time for the Lederle Laboratories in New Jersey, found an unknown
substance active against different bacteria, rickettsias and viral pathogens. He
named the substance Aureomycin, publishing his results in 1948(1) and patenting
them in 1949. (2)
In 1950, researcher from Pfizer also obtained a patent for the fermentation and the
production of a similar substance, called Terramycin. The structure of both molecules
remained unknown until 1953 when they were elucidated in a collaboration between
chemists at Pfizer and Prof. R. B. Woodward. (2b) In the same year, Conover at Pfizer
chemically modified aureomycin, thus obtaining a more stable compound that was
named tetracycline. (2c, 3)
Fig. 1.1 : Structures of Aureomycin, Terramycin and Tetracycline.
Nowadays, tetracyclines are divided into three different “generations”.
First generation comprises chlortetracycline, oxytetracycline and tetracycline itself.
Second generation tetracyclines are considered those synthesized between 1965
and 1972; (4) among others, it is obligatory to cite doxycycline and minocycline.
Finally, third generation tetracyclines are the so called glycylcyclines, a class of
compounds developed in early 90s, with the major representative being tigecycline
(Fig.1.2). (5)
Introduction
2
Fig. 1.2 : Structures of a) Minocycline, b) Doxycycline, c) Tigecycline
The bacteriostatic activity of tetracyclines is associated with a reversible inhibition of
bacterial protein synthesis. It is in fact known that tetracyclines bind reversibly to the
small 30S subunit of bacterial ribosome thus preventing the attachment of aminoacyl
- tRNA to the ribosomal acceptor. (6, 7) The weak interaction of tetracycline with 80S
ribosomes and the poor accumulation in mammalian cells explain the selective
antimicrobial activity of tetracyclines and their low toxicity.
The activity of these molecules against various protozoan parasites can be explained
by mitochondrial protein synthesis inhibition, because tetracyclines bind also to 70S
mitochondrial ribosomes. However, the tetracyclines are active also against different
mitochondria-lacking protozoa, an observation that has no molecular explanation at
present. (8)
Because of their broad-band spectrum activity and their low toxicity, tetracyclines
were extensively used since their discovery as a therapeutic agent in human and
veterinary medicine to treat various infections caused by Chlamydia, Rickettsia,
Brucellosis and Spirochete, and also utilized as growth promotors in animal
husbandry.
Introduction
3
This widespread use accelerated the diffusion of resistance among many commensal
and pathogenic bacteria. Mechanism of bacterial resistance to tetracycline antibiotics
can be subdivided into three different processes:
- Synthesis of the efflux protein TetA
- Synthesis of ribosomal protection proteins Tet(M)
- Enzymatic inactivation of tetracyclines
Tetracycline efflux is achieved by a membrane export protein that functions as an
electroneutral antiporter system which catalyzes the exchange of tetracycline-
divalent-metal-cation complex for a proton.
Ribosome protection is mediated by a soluble protein, named Tet(M), which shares
homology with the GTPases participating in protein synthesis, i.e. EF-Tu and EF-G.
The expression of Tet(M) allows bacterial cells to pursue protein synthesis also in
presence of tetracyclines.
The third mechanism involves a cytoplasmic protein that chemically modifies
tetracycline. This reaction only takes place in the presence of oxygen and NADPH
and does not function in the natural host (Bacteroides). (9)
The first two mechanisms are the most common and the genes encoding these
proteins are normally acquired via transferable plasmids and/or transposons, thus
increasing the probability of resistance spread. These two mechanisms were
observed both in aerobic and anaerobic Gram-negative or Gram-positive bacteria
demonstrating their wide distribution among the bacterial kingdom.
In Gram-negative bacteria the most common mechanism of resistance is that
mediated by TetA. Bacteria do not constitutively express this protein, because it
would be disadvantageous for the cell in the absence of [Tc-Mg]+, since TetA
interferes with the maintenance of the electrostatic potential across the cell
membrane.
Tight control for the TetA encoding gene is thus extremely important for the bacteria-
cell. This control is achieved by the bacterial cells through the expression of a DNA-
binding protein called TetR. This protein when bound to DNA inhibits the TetA
expression, but as soon as a minimal concentration of tetracycline diffuses in the cell
Introduction
4
and binds to TetR, the gene encoding TetA can be translated, increasing protein
expression and therefore tetracycline efflux (Fig.1.3).
Fig 1.3 : Tetracycline diffuses in the cell, where it complexates with Mg2+ ions. This complex binds to the TetR protein, which leaves its operator tetO, turning the transcription of genes tetR and tetA on.
The antiporter protein TetA is inserted in the membrane and expels [tc-Mg]+ out of the cell. (reproduced from Sänger et al. (ref.10))
The high affinity of TetR protein to the DNA operators tetO1 and tetO2, together with
the high affinity binding of its inducers, tetracyclines, explain why this system is
utilized in molecular biology as a tool for the regulation of gene expression in
transgenic organisms. This genetical switch was deeply investigated and widely
applied, leading to a precise knowledge of the molecular mechanisms of the
tetracyclines-TetR and TetR-DNA interactions. (11,12)
Considering all that, synthesis of novel tetracycline derivatives or analogues thereof
would be of great benefit in order not only to achieve new antibiotics, but also
effective inducers for the aforementioned gene control system.
New inducer-protein pairs could offer the possibility to study genes whose function is
still unknown, and in a future these controlled inducible expression systems could be
applied for gene therapy.
Background and Aims
5
2. Background and Aims The aims of this work can be subdivided into three main branches:
- Synthesis of new 4-dedimethylamino doxycycline derivatives as inducers of
the TetR protein
- Synthesis of new doxycycline derivatives for applications in Surface Plasmon
Resonance technology
- Development of a click chemistry approach for the chemical modifications and
conjugation of doxycycline.
2.1 Synthesis of new 4-dedimethylamino doxycycline derivatives as inducers of TetR protein
The tetracycline responsive regulatory systems have been widely applied to control
gene activities in eukaryotes providing a precisely regulated control of transgenic
expression that is reversible, quantitative and reproducible. These systems are even
commercially available (TET Systems Holding, Clonetech) and they were shown to
function in cultured cells from mammals, plants, amphibians and insects as well as in
whole organisms including yeast, Drosophila, plants, mice and rats. (1, 2, 3)
Since its first report in 1992 from Gossen and Bujard, (4) the Tet system
revolutionized the possibility to study gene functions in vitro and in vivo. Two
established systems are called Tet-on and Tet-Off. In the Tet-Off system, based on
rtTA protein, gene expression is turned on when tetracycline (Tc) or doxycycline
(Dox) is removed from the culture medium. In contrast, expression is turned on in the
Tet-On system (based on the tTA protein) by the addition of doxycycline. Both
systems permit gene expression to be tightly regulated in response to varying
concentrations of tetracycline or doxycycline (Figure 2.1.1).
Both tTA and rtTA are fusion proteins, consisting of wild type tetR (tTA) or a 4 amino
acids mutant tetR (rtTA)(5) fused together with VP16, a transcription activator protein
derived from herpes simplex virus.(6) It is important to remark the fact that, in contrast
to what happens in nature for tetracycline resistance phenomenons, in these systems
transcription is activated when tetR derived proteins bind to their operator tetO, which
is inserted near the gene of interest.
Background and Aims
6
Fig. 2.1.1 : For details see text. (figure taken from ref. 4)
To increase the selectivity of the Tet system, it is of great interest to have new
inducer-repressor pair that could switch a gene in an on-off way. Together with the
group of Prof. Hillen of the microbiology department at University of Erlangen-
Nürnberg, this could be achieved from one side synthesizing novel tetracycline
derivatives, from the other creating new mutants of the TetR protein. Even more
interesting would be if the new molecules will lack an antibiotic activity. Final scope of
the investigation is to obtain protein mutants selectively responsive to new, non
antibiotic derivatives.
The chemical modifications on the doxycycline core should be rationally directed
towards sites of the molecule that tolerate variations. This can be done because
extensive studies on tetracycline have already mapped clear structure activity
relationships, summarized in fig 2.1.2.
Background and Aims
7
Fig. 2.1.2 : The minimal core still capable of antibiotic activity is drawn. Fundamental is the
stereochemistry of the fusion between rings A with B and B with C. The blue bracket shows the upper peripheral modifiable region. The red bracket shows the lower peripheral nonmodifiable region and the nonmodifiable C3-C4 region. Group in position 2 is usually modified to achieve pro-drugs derivatives.
Aim of the work was to synthesize new derivatives starting from doxycycline, one of
the best TetR inducers, firstly eliminating its antibiotic activity, removing the
dimethylamino group in position 4, and then to modify it in position 9 (fig 2.1.3).
The introduction of different substituents should lead to a comprehension in how
different functionalities would influence the binding and the induction properties of the
molecule.
Fig. 2.1.3 : Synthesis and modifications of 4-DDMA-Doxycycline.
Background and Aims
8
2.2 Synthesis of new doxycycline derivatives for applications in Surface Plasmon Resonance technology
Biosensors are devices consisting of a biological part (e.g. DNA, protein, cell,
enzyme) and a physical transducer (semiconductor, electrode, optical component).
For the application in drug discovery, they should allow the screening of a broad
variety of compounds from different sources with a reasonable throughput.
Research and development in biosensors lead to many experimental or commercial
systems on different biological levels (cell, membranes, proteins) and detection
principles (electrochemical, optical). (7,8,9)
Surface plasmon resonance-based instruments are nowadays the most popular class
of biosensors. Their label-free detection, the real-time data acquisition possibilities,
their high degree of automation and throughput, as well as the ease of use made
them to a valuable tool in drug discovery. An extremely wide range of molecules can
be analyzed, from small drugs, DNA, peptides, or proteins up to virus particles or
even whole cells. Compared to classical endpoint assays, which are mainly based on
competition or inhibition experiments, SPR sensors provide much more information
and properties simultaneously, such as ligand-protein, protein-protein interaction and
calculation of association / dissociation constants. (10,11)
Recently, a collaboration between the groups of Prof. Hillen in Erlangen and Prof.
Petz in Wuppertal developed a new strategy for the analysis of tetracycline residues
in foodstuffs. They designed a biosensor assay based on SPR for tetracycline
residues in foodstuffs, taking advantage of the most important resistance mechanism
against tetracycline in gram-negative bacteria (tetA protein).
They immobilized the operator tetO to the chip. Then, a solution of TetR was
injected, binding steadly to his operator. If a sample containing a tetracycline is
injected, the complex tetO-TetR is dissolved, giving a signal change that is registered
by the system. Using this system, the authors were able to measure tetracycline
residues in concentrations corresponding to the maximum residue limit (MRL) set by
the European Union. (12)
Background and Aims
9
Our intention was to elaborate a system just with the contrary approach, namely
synthesizing new doxycycline derivatives that could be bound to the sensor chip.
The concept is depicted in fig 2.2.1. A doxycycline derivative is immobilized to the
sensing surface of the sensor chip; then, a solution of TetR protein is injected in the
system and the protein binds to the derivative, giving a change in the refractive index
signal. If a tetracycline is introduced, it should compete with the bound derivative for
the binding site of TetR, removing the protein from the system, where the bound
derivative remains. The system should register the biological event with a tetracycline
concentration – dependant signal change.
Fig. 2.2.1 : Strategy for a SPR based assay of tetracycline residues.
The majority of commercial available sensor chips present a carboxylic acid (-COOH)
group for the immobilization of the biological entity of interest (molecule, peptide,
antibody), that can be coupled via an amide bond. Doxycycline had then to be
modified with the introduction of an amino group directed towards the exit of TetR
binding domain, since the core of the molecule should still be able to bind the protein.
This amino functionality should be connected to the molecule’s core with linkers
differing in length. Two possible strategies are represented in fig 2.2.2.
The strategy drawn on the left side, plains the insertion of Boc-protected amino acids
via peptide coupling with 9-amino-doxycycline, with consequent removal of the
protecting group to afford the primary amine.
The strategy on the right side counts on the assembly of the goal structure using
cross-coupling reactions, starting from 9-halo-doxycycline.
Background and Aims
10
Fig. 2.2.2 : Strategies for the introduction of an amino linker into doxycycline core.
Background and Aims
11
2.3 Development of a click chemistry approach for the chemical modifications and conjugation of doxycycline
Nature is an excellent chemist. It synthesizes an enormous quantity of well diversified
molecules using a modular combination of little building blocks, linking them in the
majority through carbon – heteroatom bond. Inspired by nature’s combinatorial
approach, in 2001 Sharpless et al. described the necessity of a new strategy for
organic synthesis, coining the name “click chemistry” to refer to this guiding principle.
Click chemistry approach should lead to the synthesis of drug-like molecules
accelerating the drug discovery process by utilizing a few practical and reliable
reactions. (14,15,16,17)
Among all possible “click” reactions, one proved itself as the perfect example, up to
becoming simply “the” click reaction: the copper catalyzed Huisgen 1,3-dipolar
cycloaddition of alkynes with azides to form 1,4-disubsituted-1,2,3-triazoles (fig.
2.3.1).
Fig. 2.3.1 : The Click Reaction.
The application of the click approach to tetracycline research is of particular interest.
From one side, the modularity of this kind of reactions permit a parallelization that
would bring to the introduction of the most disparate chemical moieties, using the
same reaction conditions. The diversification of the lead structure is a fundamental
principle to obtain reliable SAR guidelines.
Therefore the click chemistry approach reveals very appetizing since chemical
modification of tetracyclines proved to be difficult and limited to few reactions,
because of their sensitive structure.
On the other hand, this concept revealed to be suitable for bioconjugation, as already
established by various authors (see references 16 and 17 and articles cited therein).
Background and Aims
12
To adopt the click strategy in tetracycline research, the aim of the work was to
synthesize molecules presenting an azide or an alkyne moiety. It was decided to
insert these functionalities using adequate anhydrides to be coupled to 9-amino-
doxycline. The modified tetracyclines have then to be “clicked” to various building
blocks, to obtain new triazole linked molecules (fig 2.3.2).
Fig. 2.3.1 : Strategy for the click chemistry approach to doxycycline modification.
Synthesis of Doxycycline – Peptide Conjugates
The synthesis of doxycycline-peptide conjugates is part of the goals of this project.
The interest in this conjugates is due to our collaboration with the group of Prof.
Hillen, aiming to find an alternative to the transactivaction system used nowadays. As
depicted in figure 2.3.2, in eukaryotic cells a repressor-based system is used for
gene regulation, where a fusion protein between tetR and the activating peptide
VP16 is the key entity for the transcription control. The alternative would be a system
where the transcription activating sequence is attached to the ligand, in this case
doxycycline.
Background and Aims
13
Fig 2.3.2 : Schematic representation of the reverse tTA system. A : Repressor-based; B : Ligand-
based.
Another fascinating possibility is the conjugation of peptides which could contrast the
transactivation activity of the VP16 domain, recruiting for example transcription
inhibitors through protein-protein interaction. Candidates for these derivatives are
peptides containing the motif WPRW, which are proved recruiters of inhibiting
peptides named “Groucho”. Synthesis of these conjugates should be carried out
applying the advantages of click chemistry (fig 2.3.3). (18,19)
Fig 2.3.3 : Representation of the planned doxycycline-peptide conjugates.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
14
1. Synthesis of new 4-Dedimethylamino Doxycycline Derivatives X-ray crystallography and molecular dynamics studies helped to figure out the
structural and dynamic differences that may be responsible for the induction
mechanism of TetR protein. (1-3) The atomic model thus obtained shows precisely the
binding mode of the tetracycline-Mg complex to the repressor protein and permits to
predict which part of the molecule can be further modified, without attempting its
capacity to interact with TetR.
As stated by Hillen et al. “only substitutions in positions that are in hydrophobic
contact to side-chain atoms of the protein are tolerated, namely those involving
positions 5 to 9 of tetracyclines. All other substitutions lead to either weak or inactive
antibiotics (...)”.
If we focus on the conformation that tetracyclines adopt when binding to TetR, it can
be seen that they enter the binding tunnel directing their ring A functionalities forming
hydrogen bonds with three well-conserved amino acid residues, His64, Asn82 and
Gln116, and anchoring their lower part (the 1,3 keto-enol system of oxygens at C11-
C12) through one magnesium ion to residues His100 and Thr103 on alpha6 helix of a
TetR monomer, and residue Glu147’ on alpha8 helix of the second monomer.
All representations clearly show that protein folding leaves an open space region on
the tunnel entrance around position 9 of the inducer (fig.3.1). (4)
Fig 3.1 : Chlortetracycline interactions with TetR protein.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
15
Extensive structure activity relationship rules have been deduced for tetracyclines;
concerning inducing activity, one of the fundamental regions for this antibiotics family
is position 4. In fact, epimerization at position 4 results in about 300-fold reduced
binding and 80-fold reduced induction. Substitution of this group by hydrogen in 4-
dedimethylamino-tetracycline results in no binding and no induction. (5)
4-dedimethylamino-tetracycline derivatives lack therefore not only antimicrobial
activity, but they are also no more capable to act as inducers for TetR family proteins.
Various studies of the group of Prof. Hillen demonstrated the possibility to teach TetR
to recognize new inducers, including molecules lacking the dimethylamino group in
position 4. Employing a directed evolution approach to screen appropriate TetR
mutants, they constructed a mutant protein (H64K S135L) responsive to the
derivative cmt-3 (4-dedimethylamino-6-demethyl-6-deoxytetracycline). Responsivity
was imputed particularly to mutation in position 135. (6)
In a second work, random mutations of this double mutant protein were directed to
the residues at positions 82 and 138, yielding a TetR mutant (H64K S135L S138I)
with specificity for the tetracycline analogue 4-dedimethylamino-anhydrotetracycline
(4-ddma-atc). (7)
Based on these findings the goal of the work was to synthesize 4-dedimethylamino
doxycycline derivatives bearing additional substituents in position 9, and test them for
their induction and binding activity towards selected TetR mutants. Moreover, it was
planned to perform a mutant screening in order to find new selective inducer-protein
mutant pairs.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
16
3.1 4-Dedimethylamino-Doxycycline Derivatives (DDMA-Dox) 4-Dedimethylaminotetracycline derivatives are also known as CMTs (Chemically
Modified Tetracyclines). This class of molecules lacks antibacterial activity but is
nonetheless studied for the treatment of a variety of diseases (for example as
inhibitors of matrix metalloproteinase(8)). Their synthesis was first described in a
patent dating 1962. (9)
3.1.1 Synthesis of 4-Dedimethylamino Doxycycline
Starting from doxycycline (1), the respective dedimethylamino derivative 3, also
known as CMT-8, can be synthesized in 2 steps: at first, the dimethylamino group is
methylated with CH3I to give doxycycline methiodide (2); the second step involves
the reductive elimination of the quaternary amino group with zinc dust in 50%
aqueous acetic acid (scheme 3.1.1a).
Scheme 3.1.1a : Synthesis of 4-dedimethylamino doxycycline (3).
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
17
3.1.2 Substitutions in Position 9 Synthesis of Reactive Intermediates
Starting from 4-dedimethylamino doxycycline (3), substitution in position 9 was
focused on groups that could be then further modified. Of particular interest are
compound like 9-amino and 9-halogen (Br, I) 4-dedimethylamino doxycycline.
9-Amino Derivative
For the synthesis of 9-amino, 4-dedimethylamino doxycycline (5), the same method
described in literature for doxycycline was used.(10,11) 4-DDMA-Dox (3) was first
nitrated at 0°C with potassium nitrate in concentrated sulphuric acid. Afterwards, the
aromatic nitro group was selectively reduced to the amine with H2 in presence of a
palladium catalyst. Finally, the compound is purified via reverse phase MPLC to
separate it from the main by-product, the 7-amino derivative (scheme 3.1.2a).
Scheme 3.1.2a : Synthesis of 9-amino-4-dedimethylamino-doxycycline.
9-Halogen Derivatives
The attempts to obtain 9-halogen-4-DDMA-Doxycycline showed to be more
strenuous than expected. The selective insertion of bromine in position 9 was
particularly challenging, with different reaction conditions leading to complete
different chemoselectivity of bromine addition. As shown in scheme 3.1.2b, the
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
18
reaction of compound 3 with Br2 and acetic acid led not to completeness, thus being
not particularly intriguing.
As alternative a different reactant for the bromination was investigated: N-
bromosuccinimide. Employing chloroform as solvent, it was possible to achieve a
monosubstitution in the doxycycline core, according to LC-MS analysis. Surprisingly,
bromine was not inserted in position 9, but in position 11a, as confirmed by 1H and 13C-NMR spectroscopy (compound 10). Changing solvent from chloroform to
trifluoroacetic acid (TFA), acting also as a acid catalyst, permits to achieve a di-
bromo-substitution. Spectroscopic analysis show that substitutions were directed in
position 9 and position 11a (compound 9).
Treatment of this compound with sodium dithionite affords finally the desired 9-
bromo-4-DDMA-doxycycline (8). (12,13,14)
Scheme 3.1.2b : Synthesis of 9-bromo-4-dedimethylamino-doxycycline
Much more reliable is instead the iodination of the derivative 3. In fact, the reaction
with N-iodosuccinimide in TFA proceeds smoothly and with a high regioselectivity.
First attempts conducted with H2SO4 as acid catalyst gave a mixture of 7 and 9
substituted 4-DDMA-doxycycline in a 1:1 ratio. Changing from H2SO4 to
trifluoroacetic acid led to an increase in the ratio to 5:1 in favour of position 9.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
19
Pure 9-substituted 4-DDMA Dox (6) could be afforded after reverse phase medium
pressure liquid chromatography (MPLC).
In addition, this derivative could be further iodinated in position 7 with an additional
equivalent of NIS. The 7,9-diiodo derivative (7) offers the possibility of chemical
decoration in two different positions both in the peripheral modifiable region (scheme
3.1.2c).(15)
Scheme 3.1.2c : Synthesis of 9-iodo- and 7,9-diiodo-4-ddma doxycyline.
Considering the easier insertion and the higher reactivity of the iodo derivative, the 9-
bromo derivative was discarded for the investigations of cross-coupling reactions.
3.1.3 Further modifications of 4-Dedimethylamino Doxycycline The most successful tetracycline derivative in the last 30 years was tigecycline,
which entered the market in 2005. It was thus rational to refer to it as new lead
structure for the development of structural analogues.
Tigecycline posses the central core of minocycline, and differs from this for the
substitution in its position 9. Analyzing its molecular structure carefully, it is possible
to subdivide the substituent in position 9 into three moieties with different
characteristics: an amide functionality, directly bond to ring D of the core; a basic
secondary amine moiety; and a tert-butyl rest, a particularly sterically demanding
group (figure 3.1.3a).
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
20
Fig. 3.1.3a : Structural analysis of tigecycline.
Taking advantage of this optimized molecule, it was of interest to synthesize similar
analogues starting from 4-dedimethylamino-doxycycline, disrupting and/or combining
these different moieties. The derivatives thus obtained could bring to a better
understanding of the contribution of each group to the pharmacological activity.
9-tert-Butyl-4-Dedimethylamino Doxycycline
As first compound of the series, it was my intention to synthesize a compound
bearing the tert-butyl rest, also present in the lead compound, but lacking the amide
and the amine functionality.
The goal compound could be easily synthesized via Friedel-Craft alkylation
dissolving the compound 3 in tert-butanol and adding methanesulphonic acid as a
Broensted acid catalyst.(16) The reaction proceeds at room temperature overnight and
the product 11 is obtained in good yields after purification via RP-HPLC (scheme
3.1.2d).
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
21
Scheme 3.1.2d : Synthesis of 9-tert-butyl-4-ddma-doxycycline (11).
9-Amino-4-Dedimethylamino Doxycycline: N-Acyl Compounds
In a second series of compounds, we wanted to retain the aryl amide moiety, with or
without the presence of sterically hindering groups.
Thus four different derivatives were planned. Acylation with acetic anhydride could
afford an analogue bearing only the minimal carboxamide functionality. Further
substitution should be addressed to compound with an increasing size of the acyl
rest, up to synthesizing an analogue bearing the amide functionality and the t-butyl
group.
Starting from 9-amino-4-ddma-doxycycline 5, the acylamido derivatives 12-14 were
smoothly synthesized by coupling with commercially available anhydrides in a DMF
solution containing NaHCO3 at room temperature for 1-4 h.
First attempts to couple 9-amino-4-ddma doxycyline with pivalic acid, utilizing the
classic peptide coupling reagent HATU to convert it to the activated OAt ester,(17) did
not afford the amide 15. The reactant was replaced with the more reactive
trimethylacetyl chloride. Surprisingly, coupling with 5 in DMF as solvent did not yield
the desired product, but a compound with a difference in mass of +55 from the
starting material, suggesting a possible condensation reaction between the substrate
and the solvent. Changing the solvent from DMF to N-methyl-2-pyrrolidinone (NMP),
as alternative polar aprotic solvent, the desired pivalamide derivative (15) could be
obtained.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
22
The reactivity of 9-amino,4-dedimethylamino doxycycline differentiate thus itself from
that of other tetracycline compounds, which are able to react successfully also with
active carboxylic acid esters.(18)
Scheme 3.1.3a : Synthesis of N-acylated derivatives of 9-amino-4-ddma doxycycline.
9-Amino-4-Dedimethylamino Doxycycline: N-Alkyl Compounds
The last functionality to be investigated was the basic amine present in the lead
structure. The insertion of this moiety into tetracycline molecules looks quite
interesting. In fact, it is possible to proceed with the modification using two different
strategies: a) starting from 9-amino-4-ddma doxycycline and alkylating the arylamine
via reductive amination; or b) modifying 4-ddma doxycycline to obtain an
aminomethylated derivative, and then decorating the amine functionality via reductive
amination (scheme 3.1.3b).
Scheme 3.1.3b : Strategies for the investigation of derivatives bearing a basic amine moiety.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
23
Obviously the second strategy would be more desirable, because in contrast to the
arylamino derivatives, the basicity of the 9-aminomethyl compounds should be more
similar to that of our lead structure.
The amino- and imido-methylation of tetracyclines are reactions that were carried out
already at the early stage of tetracycline research.(19,20) It was only in 2002 that these
kinds of reactions were investigated for the achievement of 9-aminomethyl-
tetracycline.(21) Researcher of the company Paratek applied for a patent in which they
describe the preparation of a 9-aminomethyl derivative of minocycline, the structural
core of tigecycline. They proceeded in a three steps synthesis utilizing N-
hydroxymethylphtalimide as reagent for the aminomethyl synthon (scheme 3.1.3c).
One derived compound, designated PTK 0796, has been chosen for development
and is currently in Phase I human clinical trials.
Scheme 3.1.3c : Synthesis of 9-aminomethyl minocycline.
The same reaction procedures were applied to obtain a 9-aminomethyl derivative of
4-dedimethylamino doxycycline, but all attempts were unsuccessful (scheme 3.1.3d).
In point of fact, the first step of the synthesis plan was successful and a 2,9-bis-
aminomethylphtalimido-4-ddma doxycycline was obtained. The removal of the
phtalimido protecting group and of the aminomethyl group in position 2 were instead
unachievable, also when trying to use alternative conditions.(22)
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
24
Scheme 3.1.3d : Synthesis of 9-aminomethyl, 4-ddma-doxycycline.
The other strategy was thus to be exploited. Aldehydes and ketones react with
amines via reductive amination giving N-alkylated compounds. This reaction offers
the possibility to insert a variety of alkyl chains to study the influence of the steric
effects of the derivatives in terms of binding and induction activity to TetR.
Taking advantage of the potential that this reaction gives, it was decided to
synthesize mono- and di-alkylated compounds, derivatives decorated with branched
alkyl chains, and with cyclic alkyl chains.
To obtain mono- and di-alkylated derivatives, the reaction of 9-amino-4-ddma
doxycycline (5) with different aldehydes was investigated.
No reaction condition was found to be suited in order to obtain mono-substituted
derivatives. In every case, a mixture of the mono- and di-substituted alkyl derivatives
was obtained, also when using a stoichiometric equivalent or less of aldehyde, or
lowering the reaction temperature. The investigation of an alternative reducing agent
(NaBH(OAc)3) was also unsuccessful.
Starting from compound 5, reaction with formaldehyde, acetaldehyde and
propionaldehyde afforded respectively 9-dimethylamino, 9-diethylamino and 9-
dipropylamino 4-dedimethylamino doxycycline (compounds 16, 17 and 18) (scheme
3.1.3d).
Reaction is afforded via imino-formation and subsequent reduction with sodium
cyanoborohydride. This mild reducing agent assures the chemoselective reduction of
imines without attempting to other sensitive groups present in the molecules, such as
ketones of the keto-enol systems.(23)
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
25
Scheme 3.1.3d : Synthesis of 9-di-alkyl-4-ddma doxycyclines.
As for the achievement of branched alkyl chains and cyclic alkyl derivatives,
compound 5 could be reacted with ketones. In this way, reaction with acetone,
cyclopentanone and cyclohexanone afforded compounds 19 (9-isopropylamino), 20
(9-cyclopentylamino) and 21 (9-cyclohexylamino) (scheme 3.1.3e).
Scheme 3.1.3f : Synthesis of branched and cyclic alkyl derivatives starting from 9-amino-4-ddma doxycycline.
It is worth noticing that, conversely to what happened with aldehydes, the reaction of
9-amino-4ddma doxycycline with ketones, also in presence of a stoichiometric
excess of the reactant, yielded only monosubstitution products.
To exploit this reaction at a greater extend, 9-isopropylamino derivative 19 was
further alkylated; its reaction with formaldehyde afforded compound 22 (N-
isopropyl,N-methylamino) (scheme 3.1.3g), a molecule with branched alkyl moieties.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
26
This modification looks particularly interesting considering the possibility to decorate
the aromatic amino functionality with diverse alkyl chains.
Scheme 3.1.3g : Synthesis of 9-N-Isopropyl,N-methyl-amino-4-dedimethylamino doxycycline.
3.1.4 Further modifications of 9-Iodo-4-Dedimethylamino Doxycycline Palladium catalyzed cross-coupling reactions are well-known and reliable carbon-
carbon and C-heteroatom bonding reactions. Their synthetical power and their
tolerance toward a large number of functional groups facilitated their widespread use
and their assertion to prominent processes in organic synthesis. Aryl halides and aryl
triflates are the coupling partners for different substrates and usually these reactions
are named after their principal investigators.(24)
Considering the fact that this kind of chemical transformations were even applied for
total synthesis of natural products, commonly sensitive substrates, it was decided to
investigate the applicability of such reactions for the chemical modification of 4-
dedimethylamino doxycycline, and more precisely the Suzuki, Sonogashira and
Buchwald-Hartwig cross-coupling reactions.
Sonogashira Coupling
In a paper dated 2003,(25) Nelson et al. described the possibility of reacting diazo- and
iodo- modified tetracyclines with alkenes and alkynes to afford new Heck, Suzuki and
Sonogashira type derivatives.
Applying the same conditions used by the authors for the reaction between
minocycline or sancycline and alkynyl reactants, it was not possible to afford
Sonogashira coupling products. After few optimization steps, I could establish a
reliable method for obtaining such derivatives starting from 9-iodo-4-dedimethylamino
doxycycline. The reaction of 6 with phenylacetylene, 1,7 octadiyne and N-(5-hexynyl)
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
27
phthalimide afforded molecules 23 (9-Phenylethynyl), 25 (9-Octa 1’,7’diynyl) and the
phtalimido derivative 26 respectively (scheme 3.1.4a).
Scheme 3.1.4a : Synthesis of Sonogashira derivatives starting from 9-iodo-4-dedimethylamino doxycycline.
At the contrary, the reaction of 9-iodo derivative 6 with propargylamine was
unsuccessful. A certain conversion of the starting material could be observed but in
very little percentage. Moreover, LC-chromatogram showed a double-peak
corresponding to the product mass, indexing a possible side reaction (scheme
3.1.4b, above).
For another derivative the Sonogashira reaction was disappointing. When trying to
react 9-iodo-4-dedimethylamino doxycycline with 5-hexynoic acid, the desired linear
compound 24a could not be obtained. Instead, the benzofuran derivative 24b, a
product of the ring closure between position 9 and 10 was formed. This was probably
due to the fact that at room temperature no conversion of 6 into 24a was observed,
and the reaction was then carried out at 80°C to accelerate its course (scheme
3.1.4b, below).
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
28
Scheme 3.1.4b : Reactions of 9-iodo-4-dedimethylamino doxycline with propargylamine and 5-hexynoic acid.
This side reaction could have been also predicted, because in the literature there are
lot of examples for the application of such a cross-coupling reaction for the synthesis
of benzofuranes and other ring systems.(26)
Further Derivatization of 9-Iodo-4-Dedimethylamino Doxycycline (II)
The successful application of Sonogashira reaction offers an alternative approach for
the achievement of a 9-aminomethyl derivative. In fact, as shown by the
retrosynthetic scheme 3.1.4c, nitriles are synthons for this moiety, and cyanide salts
can be introduced into molecules utilizing the Sonogashira cross-coupling reaction.
(27)
Scheme 3.1.4c : Retrosynthetic analys for 9-aminomethyl-4-dedimethylamino doxycycline.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
29
Reacting compound 6 with potassium cyanide in presence of tetrakis
triphenylphosphine palladium(0) lead to cyano compound 28 (scheme 3.1.4d).
Scheme 3.1.4d : Synthesis of 9-cyano-4-dedimethylamino doxycycline.
The traditional way to reduce a nitrile is using metal hydrides, such as LiAlH, that
would surely lead to decomposition of fundamental groups of the tetracycline core.
Trying to solve the problem of chemoselectivity, it was attempted to reduce the nitrile
group using the same conditions adopted for the reduction of nitro compound to
amine, i.e. Pd/C and H2 under high pressure, reaction occasionally used for the
reduction of nitriles, but no aminomethyl derivative could be obtained (scheme
3.1.4e).
Scheme 3.1.4e : Attempt to reduce 9-cyano-4-dedimethylamino doxycycline into 9-aminomethyl
derivative.
Another interesting chemical transformation of nitriles undergo is the [2+3]
cycloaddition with azide to yield 1H-tetrazoles. This functionality is usually considered
as a bioisoster of the carboxylic acid group.
In order to obtain such a derivative, two different conditions were tried: firstly, the
cycloaddition of sodium azide in presence of ammonium chloride with conventional
heating (100°C) or under microwaves irradiation, obtaining no conversion at all. In a
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
30
second attempt, ammonium chloride was replaced by zinc bromide, and the solvent
was changed from DMF to a water/2-propanol mixture, as described by Sharpless et
al.(28) Also in this case, both classical heating and microwaves irradiation as source
for thermal activation were unsuccessful (scheme 3.1.4f).
Scheme 3.1.4f : Experiments for the formation of a 9-tetrazol-4-dedimethylamino doxycycline.
Suzuki Coupling
In the aforementioned publication,(25) some examples of tetracyclines modified via the
Suzuki-Miyaura reaction are described. Since the authors were dealing with 9-diazo
derivatives, their reaction conditions had to be adapted in order to be successfully
applied to 9-iodo-4-dedimtheylamino doxycycline. After few optimization steps a
reliable methodology could be accomplished.
Initially, reactions were carried out in a solvent mixture consisting of MeOH, DMF and
H2O, in the presence of Na2CO3 as base and Pd(OAc)2 as catalyst, at 80°C for 4-5h.
Reacting 9-iodo derivative 6 with phenylboronic acid gave compound 31 (9-phenyl-4-
ddma doxycycline) while the reaction with 4-carboxyphenylboronic acid afforded 9-(p-
carboxyphenyl)-4-dedimethylamino doxycycline (32)(Scheme 3.1.4g).
Both reactions were then investigated utilizing microwave irradiation. In this case,
they can be carried out without particular attention of an inert atmosphere. Moreover,
all reactants could be directly put in the mixture, and there was no necessity of a
solvent mixture, since the only solvent used was DMF. After only ten minutes of MW
irradiation at 100°C (average 80W), HPLC-MS analysis show complete conversion of
starting material into products.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
31
Scheme 3.1.4g : Synthesis of compounds 31 and 32 via palladium catalyzed Suzuki-Miyaura
reaction.
Buchwald-Harwtig Coupling
As an alternative to get compounds substituted in position 9 with N-alkyl groups via
reductive amination, as described in paragraph 3.1.3, it was decided to investigate
the reactivity of 9-Iodo, 4-dedimethylamino-doxycycline in the so called “Buchwald-
Hartwig” cross-coupling reaction.
Differently from the successfully applied Suzuki and Sonogashira coupling reactions,
which offer the possibility to form new C-C bond, the scope of the Buchwald-Hartwig
reaction is the formation of new C-N bonds. This is usually accomplished using a
palladium catalyst in presence of a ligand and a base (29).
All attempts to apply this coupling reaction with the 9-iodo modified doxycycline were
unsuccessful. A first try was conducted with benzylamine, using palladium acetate as
catalyst and cesium carbonate as base, and BINAP as ligand, classic conditions for
this coupling reaction. No product could be observed at 80°C even after 24 hours
(scheme 3.1.4h).
Scheme 3.1.4h : Buchwald-Hartwig coupling reaction with compound 6.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
32
In 2001 Buchwald itself (30) proposed alternative conditions for the amination and
amidation of aryl halides using a copper catalyst in the presence of a diamine ligand.
They described these conditions as “an enhanced version of the Goldberg reaction” (31). Unfortunately, also using these conditions it was not possible to obtain some
conversion of compound 6 into the desired products (scheme 3.1.4i).
Scheme 3.1.4i : Amination and amidation trials on compound 6 using a modified Goldberg-reaction.
Another methodology for this kind of reaction was developed by the group of Prof.
Ma.(32,33,34) They noticed the accelerating properties of α amino acids in the outcome
of the same, showing a yield increase when a catalytic amount of L-proline was used,
and obtaining coupled products starting from both electron-rich and electron-poor aryl
iodides.
Again, the application of this promising methodology failed to give products when
applied to our 9-iodo derivative (scheme 3.1.4j).
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
33
Scheme 3.1.4j : Attempts to obtain amination of compound 6 with conditions developed by Prof. Ma.
Reactivity Studies on 7,9-Diiodo-4-Dedimethylamino Doxycycline.
As reported in paragraph 3, starting from iodo compound 6 it was possible to obtain a
7,9 diiodo substituted 4-dedimethylamino doxycycline (7). Using palladium chemistry,
this molecule offers the possibility of being modified in two positions both belonging
to the upper modifiable region. It was then logical to study its reactivity utilizing the
reactions that had been already successfully applied for the 9-iodo derivative, i.e. the
Sonogashira and the Suzuki reactions.
Concerning the Sonogashira derivatization, reaction of compound 7 with one
equivalent of phenylacetylene, using the same conditions developed for 9-iodo-4-
DDMA doxycycline, afforded the product 29 (7-Iodo-9-phenylethynyl-4-
dedimethylamino doxycycline). As confirmed by HMBC-NMR studies, the coupling of
the phenylacetylene to the doxycycline derivative occurs only at position 9. The
achievement of such a regioselective product makes this reaction interesting,
because of the possibility to differently modify the two positions with diverse
acetetylenes.
Reacting compound 7 with an excess of reactant, yields a complete conversion into
the 7,9-bis-phenylethynyl-4-dedimethylamino doxycycline 28 (scheme 3.1.4k).
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
34
OH O OH O
OH
O
NH2
CH3 OH
OH OH O OH O
OH
CH3 OH
OHCONH2I
7
OH O OH O
OH
CH3 OH
OHCONH2
30
I I
29
phenylacetylene (1eq),TEA, THF, rt, 3h
Pd[(Ph)3P]4 10% CuI 10%
Pd[(Ph)3P]4 10% CuI 10%
phenylacetylene (3eq),TEA, THF, rt, 3h
Scheme 3.1.4k : Sonogashira reactions with 7,9-diiodo-4-dedimethylaminodoxycycline.
On the contrary, the reaction of 7 under Suzuki conditions already used for the
derivatization of compound 6 showed no chemoselectivity at all. In fact, reacting it
with an equimolar amount of phenylboronic acid afforded a mixture of 7-phenyl and
9-phenyl doxycycline derivative, which could not be separated chromatographically.
The reaction with an excess of the reactant yielded easily the 7,9 bis-phenyl
derivative 33 (scheme 3.1.4l).
Scheme 3.1.4l : Suzuki-Miyuara reactions with 7,9-diiodo-4-dedimethylaminodoxycycline.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
35
3.2 Biological Investigations. In vivo inductions properties and antibiotic activity of selected derivatives
Selected derivatives were tested in the group of Prof. Hillen for their antibiotic activity
and their induction of different TetR mutants. Table 3.2a presents the data thus
obtained.
MIC (mg/mL)
MIC(tetO) E.coli LacZ (% B-Gal) Name
WH207 NR698 B. subtilis WH207 NR698 TetR
(BD) S135L i2 r2
dox 2 1-2 0. 5 32 8 83.3 81 1.7 2.7
3 32 2 0.5 32-64 2 1.7 46.2 70.1 89.0
5 >64 nd nd >64 nd 1.9 1.3 29.9 93.0
11 >64 1 1-2 64 2 0.8 0.9 3.3 78.6
12 >64 >64 >64 >64 >64 1.2 1.1 2.1 76.3
13 >64 64 >64 >64 64 0.8 1.3 3.9 102.8
14 >64 4 8 >64 4 1.2 1.5 18.8 91.2
15 >64 4 8 >64 4 1.0 1.0 1.2 99.6
17 >64 32 32 >64 16 1.0 1.0 1.2 87.5
18 >64 >64 64 >64 32-64 0.8 0.9 1.5 97.3
19 >64 4 8 >64 4 1.0 1.0 1.0 100.6
20 >64 2 4 >64 2 0.8 0.8 0.9 91.6
21 >64 4 nd >64 1 0.8 0.8 1.0 85.4
23 >64 1 2 >64 1 0.8 1.0 32.3 83.5
24b >64 64 >64 64 64 1.0 1.0 4.1 89.2
25 >64 2 4 >64 2 1.2 1.4 13.8 99.4
26 >64 nd nd >64 >64 3.2 0.8 1.2 74.4
31 >64 1 1 >64 2 0.7 0.9 5.7 62.8
Table 3.2a : MIC = Minimum Inhibitory Concentration. % B-Gal = increase of beta-galactosidase activity. WH207 = E.coli strain. NR698 = E.coli leaky mutant (increased
membrane permeability). TetR(BD) = wild type Tet repressor. TetRi2 = H64K S135L S138I mutant (4-ddma sensible). TetRr2 = E15A L17G L25V mutant (reverse TetR). TetR(S135L) =
S135L mutant (relaxed TetR).
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
36
As showed in the table, the derivatives posses no antibiotic activity for E.coli cells. As
suggested by the literature, this should be due to the lack of the 4-dedimethylamino
group. However, the results obtained with the leaky mutant NR698, an E.coli which
posses an increased membrane permeability, and with B. subtilis, a Gram positive
bacteria, open new questions.
Quite all derivatives show an antibiotic activity on these two bacteria species, even in
presence of Tet(O), the ribosomal protection protein, which mediates one of
tetracycline resistance phenomenon. The first result suggests that the antibiotic
inactivity on E. coli can probably be attributed to a reduced ability of the 4-ddma
derivatives to pass the outer bacterial wall. The “fundamental” role of the
dimethylamino group in position 4 should thus be called into question.
Their antibiotic activity in presence of Tet(O) suggest that these derivatives can exert
their action in different ways than by inhibiting the 30s ribosomal subunit.
From one side, this lack of antibiotic activity is fundamental for the development of
novel TetR effectors, since as stated in the introduction we are looking for non
antibiotic molecules that can induce TetR proteins.
From another point of view, these results suggest the possibility of designing new
antibiotics based on the 4-ddma core. The molecules do not bind effectively TetR,
avoiding in fact the resistance mechanism effect by the efflux protein TetA. Such a
class of molecules could be employed against resistant bacteria because of their
probably non-ribosomial mediated mode of action. As for the design of these
molecules, it should be focused on the introduction of groups that would improve
their diffusion capacity into the cells. An alternative strategy could be the introduction
in the core of another metal chelating group, since it is recognized the ionophoric
nature of tetracyclines (ionophores are organic compounds capable of forming lipid-
soluble complexes with metal cations).(35)
Concerning the TetR induction capacity of the derivatives, some interesting data
were obtained. First of all, none of the tested compounds showed an induction of the
wild type protein (TetR(BD)), as well of the S135L mutant, except for the base
structure 4-dedimethylamino doxycycline. No interesting activities were found for the
activity toward the reverse phenotype r2. This well correlates whit the finding that
good inducers for TetR wild type are usually strong corepressors for TetRr2.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
37
As for the triple mutant i2, four compounds showed good induction ability, i.e. 5, 14,
23 and 25 (figure 3.2a).
Figure 3.2a : Molecules active on TetR mutant i2.
The structural differences in these molecules do not permit the deduction of some
structure activity relationship trend. It is not clear if the lipophilic group present in
three of the molecule is responsive for a better binding and induction with TetR, or for
a better ability of diffusion into cells. Moreover, it is not sure if compound 23 exert its
relatively high induction by the presence of a π moiety (as could be suggested by the
activities of compounds 14 and 25), or of a long alkyl chain, as known by the
literature and by our group´s experience.
Their selectivity between TetR S135L and TetRi2 could permit their use for the
independent regulation of two distinct reporter genes.
Screening for new TetR-Inducers pairs.
With the goal of finding new TetR protein-inducer pair, as already stated in the aims,
we ran a random mutagenesis screening using diverse derivatives, belonging to the
class of the anhydrotetracycline and 4-dedimethylamino doxycycline. As for the latter,
two compounds were tested : 9-amino-4-dedimethylamino doxycycline and 9-phenyl-
4 dedimethylamino doxycycline (figure 3.1.5a). The derivatives were chosen because
they showed interesting inducing properties over the TetRi2 mutant.
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
38
Fig. 3.1.5a : Tested substances for the mutant screening.
In the process were screened mutants from three different “pools”. One pool (pool M)
was based on the TetR mutant that showed an increased affinity for 4-
dedimethylamino anhydrotetracycline, namely the TetRi2 (H64K S135L S138I). The
other two pools (pool A and D) were based on random mutations in the DNA region
coding for the repressor ´s binding pocket.
The plasmids contain in addition to the gene for the TetR protein (tetR) also a gene
coding for ampicillin resistance (apR). This construct permits the selection of only
useful bacterial cells, because the presence of ampicillin inhibits the growth of any
bacterial colony that presents a non mutated DNA.
The E. Coli cell-line used for the screening posses in genome a lacZ-gene under
TetR control. If the plasmid introduces a gene encoding for a functional TetR into the
bacteria cell, lacZ would then be repressed, and no galactosidase would be
expressed. β-galactosidase is an intracellular enzyme that cleaves the disaccharide
lactose into glucose and galactose, forming acidic metabolites. The pH indicator
present in the medium changes its colour into red, thus showing the bacterial
colonies bearing a functional TetR. Then, the coloured colonies were transferred into
new agar plates containing this time the substances under investigation plus a
control plate.
If the doxycycline derivative acts as inducer, the TetR mutant dissociates from DNA,
β-galactosidase is expressed and the colonies turn red (figure 3.1.5b).
Synthesis of new 4-Dedimethylamino Doxycycline Derivatives
39
Figure 3.1.5b : Representation of the random mutagenesis screening to find ne TetR mutants-inducer
pairs.
In total the three selected 4-dedimethylamino doxycycline derivatives were screened
against 1550 TetR mutants, of which 1050 were derived from pool A and D, and 500
derived from pool M. Nonetheless, no hit for a new inducible TetR protein - inducer
pair was found.
Synthesis of Doxycycline Derivatives for SPR Investigations
40
4. Synthesis of Doxycycline Derivatives for SPR Investigations The application of Surface Plasmon Resonance (SPR) in modern drug discovery
proved to be a very potent and promising field of investigation.
A first application of SPR technology in tetracycline research was already reported,
showing the possibility of studying tetracycline residues in foodstuffs, a promising
methodology for the development of a rapid system of analysis.
Our aim was to develop a similar system, but just with a reversed approach of what
already done, precisely binding a tetracycline molecule to the sensor chip of the SPR
system.
Commercial Sensor Chips.
A wide variety of sensor chips is commercially available, yet the most indicated for
the study of protein ligand interactions are the CM-5. As depicted in figure 4.1, they
consist of three parts: a glass base, a thin gold film and a dextran layer, linked to the
gold film via covalent gold-thiol bonds.
Fig. 4.1 : Schematic representation of a sensor chip CM-5.
The dextran chains bear multiple carboxylic acid functionalities that serve as an
anchor for the binding of the ligand (or protein) of interest. This could be achieved
either via direct coupling with an amine group, or by the active NHS (N-
hyydroxysuccinimide) ester that can react with other functional groups (figure 4.2).
Synthesis of Doxycycline Derivatives for SPR Investigations
41
Fig. 4.2 : Possible binding reactions for the linkage of a ligand to a CM-5 sensor chip.
It was decided to furnish doxycycline with an amino functionality linked to position 9.
Various chain lengths between the binding group and the molecule core should be
investigated, to study their influence on the binding between doxycycline and the tet
repressor protein.
4.1 Chemistry 4.1.1 Synthesis of Amino bearing Doxycycline Derivatives
9-Aminomethyl Doxycycline
Following a patent description by Paratek Pharmaceuticals it was tried to obtain 9-
aminomethyl,4-dedimethylamino doxycycline (paragraph 3.1.3). The same procedure
was applied to doxycycline, obtaining the same negative result. As for derivative 3,
also for doxycycline can be observed the first product of the reaction pathway, i.e. the
2,9-bis-aminomethylphthalimido doxycycline, but the two subsequent steps
(phtalimide cleavage and elimination of the modification in position 2) do not yield the
desired product (scheme 4.1.1a).
Synthesis of Doxycycline Derivatives for SPR Investigations
42
Scheme 4.1.1a : Attempt to obtain 9-aminomethyl doxycycline.
Derivatization through Sonogashira Reaction
The second strategy is based on a Sonogashira cross-coupling reaction, so at first it
was necessary to synthesize 9-iodo doxycycline (34). To obtain this compound, the
same reaction conditions as for 9-iodo, 4-dedimethylamino doxycycline were applied:
iodination with N-iodosuccinimide in presence of an acid catalyst. The reaction
proceeds smoothly giving 7-iodo doxycycline as main by-product, which can be
eliminated via HPLC, to afford pure 9-iodo doxycline (scheme 4.1.1b).
Scheme 4.1.1b : Synthesis of 9-Iodo Doxycycline.
Reaction of 34 with 6-phtalimido-1-hexyne, led to compound 36 (9-(6-(1,3-
dioxoisoindolin-2-yl)hex-1-ynyl) doxycycline). Cleavage of the phtalimido protecting
group led to an unwanted side reaction, the formation of a benzofuran ring between
position 9 and 10, resulting in a mixture of linear amine and benzofuran derivatives
that could not be separated via HPLC (scheme 4.1.1c). The same side reaction was
observed for some 4-dedimethylamino derivatives (paragraph 3.1.4).
Synthesis of Doxycycline Derivatives for SPR Investigations
43
Scheme 4.1.1c : Synthesis of an amino bearing doxycycline via Sonogashira coupling vith protected amino hexyne.
The ring closure from the alkynyl phenol to the benzofuran system is usually
accomplished, as reported in the literature, using an electrophilic cyclization. (1,2,3)
Some other report shows the tendency of this reaction to occur either thermally, or
through metal (Pd or Cu) catalyzation,(4,5) or just in presence of a base.(6) Evidently
the basic treatment of compound 35 with methylamine was sufficient to afford the
cyclized compound.
To avoid the treatment with base, I decided to couple an alkyne derivative bearing an
amino moiety protected with an acid-labile protecting group, the tert-butyl carbonyl
group (Boc).
9-iodo doxycycline was coupled with N-Boc protected propargylamine using the
developed Sonogashira methodology to afford compound 37 (9-(Boc-3-aminoprop-1-
ynyl) doxycycline). LC-MS investigation during reaction control clearly showed the
formation of a neat product. Unfortunatly, during the purification step the desired
product exhibited the tendency of forming the benzofuran ring, as confirmed by LC-
MS and 1HNMR studies, making also this pathway uninteresting for further
developments (scheme 4.1.1d).
Synthesis of Doxycycline Derivatives for SPR Investigations
44
Scheme 4.1.1d : Synthesis of 9-(3-Boc-aminoprop-1-ynyl) Doxycline.
To avoid any possibility of side reaction that could involve important regions of the
tetracycline core, it was decided to synthesize compounds starting from a reliable
reaction already successfully applied for 4-dedimethylamino derivatives: an acylation
of the arylamino functionality, this time starting from 9-amino doxycycline. This
strategy offers the possibility to couple commercially available N-protected amino
acids.
N-Acylated Derivatives
9-amino doxycycline is a compound known in the literature.(7) Its synthesis proceeds
as for the other tetracycline via aromatic nitration and reduction with hydrogen in
presence of a metal catalyst. Thus doxycycline affords nitro compound 38 via
nitration with NaNO3 in concentrated sulphuric acid, and its reduction yields 9-amino
doxycycline (39) which is then purified by MPLC to separate it from the regioisomer
7-amino doxycycline (scheme 4.1.1e).
Synthesis of Doxycycline Derivatives for SPR Investigations
45
Scheme 4.1.1e : Synthesis of 9-nitro and 9-amino doxycycline.
It was decided to acylate the arylamino group with two different Boc protected amino
acids, differing in terms of carbon chain length: Boc-glycine and Boc-5-aminovaleric
acid. However, coupling of these reagents with amino doxycycline using the
HATU/DIPEA method did not afford any product. As already noticed for 9-amino,4-
dedimethylamino doxycycline, the nucleophilicity of arylamino doxycycline derivatives
could be too low in acylation reactions with activated acid esters. To succeed with the
reaction either acid chlorides or acid anhydrides could be suitable. It was decided for
the latter, being symmetrical anhydrides of amino acids compounds of excellent
reactivity.
The symmetrical anhydrides were generated under nitrogen atmosphere using two
equivalents of Boc-protected amino acid and one equivalent of dicyclohexyl
carbodiimide (DCC) in DMF.
Coupling of the Boc-glycine anhydride and the Boc-5-aminovaleric anhydride with 9-
amino doxycycline afforded compounds 40 and 41 respectively (scheme 4.1.1f).
Scheme 4.1.1f : Synthesis of boc aminoacyl derivatives of doxycycline.
Synthesis of Doxycycline Derivatives for SPR Investigations
46
The second step involved the removal of the protecting group, which was achieved
using a 50:50 mixture of trifluoroacetic acid and dichloromethane at room
temperature for one hour. Starting from N-Boc protected intermediates 40 and 41,
compound 42 (9-(2-aminoacetamido) doxycycline) and 43 (9-(5-aminopentanamido)
doxycycline) were obtained (scheme 4.1.1g).
Nonetheless it was not possible to isolate the glycynoylamino derivative 42 in a pure
form, even when pursuing reverse phase chromatography with very slow gradient
increase.
Scheme 4.1.1g : Boc cleavage leading to products 42 and 43.
9-(5’-Amino-pentanamido) doxycycline (43) was acylated with acetic anhydride,
obtaining the acetamido derivative 43a (scheme 4.1.1h). This modification had to
mimic the functionality that would be obtained later when coupling the compound to
the sensor chip. The compound was tested in order to confirm the binding properties
to the wild-type TetR protein.
Scheme4.1.1h : Acylation of 43 with acetic anhydride.
Synthesis of Doxycycline Derivatives for SPR Investigations
47
To better succeed in the intent of developing a SPR system, it was decided to
synthesize some more analogues having a greater distance between the amino
group and the doxycycline core.
These molecules would outdistance the bound molecule from the carboxymethyl-
dextran chain, giving perhaps the possibility of a better interaction between the
doxycycline molecules and the repressor proteins.
Reaction of 9-amino doxycycline with the symmetric anhydrides formed from 8-Boc-
amino octanoic acid and 11-Boc-amino undecanoic acid afforded compounds 44 and
45 (9-(8’-Boc-amino-octanamido) doxycycline and 9-(11’-Boc-amino-undecanamido)
doxycycline, respectively). Cleavage of the protecting group with TFA/DCM yielded
9-(8-aminooctanamido)-doxycycline (46) and 9-(11-aminoundecanamido)-
doxycycline (47) (scheme 4.1.1j).
Scheme 4.1.1j : Synthesis of 9-(8-aminooctanamido)-doxycycline (46) and 9-(11-aminoundecanamido)-doxycycline (47).
Synthesis of Doxycycline Derivatives for SPR Investigations
48
As an alternative, the formation of a similar compound was investigated using a linker
with different chemical-physical characteristics, i.e. an eight atom polyethylenglycole
(Boc-11-amino-3,6,9-trioxaundecanoic acid, or mini-PEG-3) linker.
The formation of derivatives 48 and 49 followed the same procedure as before, with
the coupling via symmetric anhydride and then acidic cleavage of the protecting
group (scheme 4.1.1i). As for the glycylamino derivative, also this compound
presented problems in the purification step, and the compound was discarded.
Scheme 4.1.1i : Synthesis of pegylated doxycycline derivatives.
Three suitable derivatives were then synthesized, with a distance from the amino
group to the doxycycline core of 7, 10 and 13 atoms.
They were sent to the group of Prof. Petz of the University of Wuppertal, and tested
in a Biacore 3000 SPR biosensor.
Synthesis of Doxycycline Derivatives for SPR Investigations
49
4.2 Biological Investigations
Figure 4.1 shows the doxycycline derivatives that were synthesized for the Surface
Plasmon Resonance experiments. Each of them bears an amino functionality bound
to the doxycycline core via a carboxamide group in position 9. Modification in this
position allows the molecule to interact with the TetR protein, even if bound to the
carboxymethyl dextran chain of the sensor chip.
Fig. 4.1 : Doxycycline derivatives used for SPR investigations.
Immobilization of the derivatives
The three derivatives were successfully immobilized to the sensor chip CM-5, via an
amide coupling using EDC / NHS as reagents. The immobilization level is well
correlated with the length of the spacer, i.e IU95 > IU91 > IU58 (table 1).
Flow-cell Derivative Activation Level (RU)
Immobilization Level
1 IU58 155,9 477,3
2 IU91 169,7 1049,5
3 IU95 160,8 2362,4
Table 1 : Activation and immobilization levels of the derivatives.
Synthesis of Doxycycline Derivatives for SPR Investigations
50
Characterization of TetR Binding
After demonstrating a firm linkage of the derivatives to the chip, the binding to TetR
was investigated.
In multiple experiments, it was demonstrated that the TetR binding of the derivatives
posseses interesting properties: it is specific, stable, dependent on TetR
concentration, injections time duration, and flow-rate.
The specificity of the binding was investigated examining the response of the system
after the injection of a TetR solution with or without magnesium ions. As shown in
figure 4.2, in absence of magnesium no binding can be observed. In contrast, the
presence of magnesium induces a correlated change in the signal, supporting the
fact that we are dealing with a specific binding.
Fig. 4.2 : Injection of a TetR solution in absence and in presence of magnesium ions (IU91).
The stability of the ligand-protein interaction was demonstrated injecting a TetR / Mg
solution and observing the dissociation of the complex within one hour. The
derivatives showed a dissociation from TetR between 5,7 and 8,7 percent.
Synthesis of Doxycycline Derivatives for SPR Investigations
51
Competition experiments
To study the competition between the chip-bound derivatives and diverse
tetracyclines, two experiments were conducted. In one, a tetracycline derivative is
mixed with a TetR solution, and this mixture injected in the system. In the second
experiment the TetR solution is firstly injected, followed by the injection of a
tetracycline solution.
As for the first experiment, the system was studied with tetracyclines in concentration
from 0 to 650 ng/mL. The diagram and the table show the dependency of the TetR
binding to the tetracycline concentration. Moreover, in calibration experiments the
signal well correlates also with the spacer length, where IU95 shows better binding
properties than IU91 and IU58. The experiments were then accomplished with
different concentration of the TetR protein, showing similar correlations.
Figure 4.3 and Table 2 : Tetracycline concentration dependency of the system signal
Synthesis of Doxycycline Derivatives for SPR Investigations
52
In the second experiment, the competition between the bound derivative and a
tetracycline molecule for the protein was pursued in a displacement experiment.
Table 3 shows clearly that after an injection of tetracycline, a displacement of the
bound derivative from the protein’s binding pocket follows, permitting TetR to be
flushed away, as indicated by the signal decrease.
Derivative TetR-Binding (RU) Displacement of
TetR after TC-injection
Displacement of TetR in %
IU95 462,2 -116,0 RU 25,1
IU91 296,1 -89,7 RU 30,3
IU58 153.6 -80,9 RU 52,7
Table 3 : TetR-binding and displacement after injection of tetracycline (60 min.)
However, if the system is analyzed in a 15 minutes period, no correlation between
tetracycline concentration and signal decrease can be observed, as showed in figure
4.4. To the contrary, the injection of tetracycline solution at first increases the
response level, probably because the molecules bind to free sites in the TetR protein.
Fig. 4.4 : Displacement experiment of the system in a 15 minutes period.
Synthesis of Doxycycline Derivatives for SPR Investigations
53
Weak points of the system
The steady immobilization of the derivatives to the sensor chip, their stable and
specific binding to the TetR and the displacement of the protein with competitive
experiments proved the development of the system as successful.
Nonetheless, some blind spots are present that impede its application as a routine
analysis system. The problems arise from: a non constant baseline, a mediocre
sensitivity, a decrease of the derivative capacity to bind the TetR, a scanty
reproducibility. Moreover the displacement or bound TetR from the chip surface is
possible but happens only after very long injections times.
The robustness of the system is probably damaged by the chemical instability of
tetracycline derivatives, because all aforementioned factors are positive in a short
term use of the chip, but fail in a long term usage.
On the other side, the complicated biological events under investigation demand for
some more careful examinations. Of particular importance could be the interaction of
tetracyclines with the free binding pocket on the bound TetR dimer, but also the
intrinsic capacity of the injected molecules to displace the linked derivatives from the
protein.
All these factors have to be taken into account in order to improve the developed
system, on the basis of its promising perspectives.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
54
5. Development of a Click Chemistry Strategy for the Functionalization and Bioconjugation of Doxycycline
The advantages offered by the “click reaction” are very attractive for such difficult to
modify molecules like tetracyclines. In fact the CuAAC is an extremely
chemoselective reaction and was successfully applied for modifying highly functional
biomolecules such as polypeptides, nucleic acids or polysaccharides.(1,2,3) Moreover,
the combinatorial scope of this reaction has been already widely applied in drug
discovery, making it a potent tool for the synthesis of diverse “clicked” libraries.(4,5,6)
This wide application in different research areas acknowledged the click reaction as
robust and reliable, so it was decided to investigate the possibility of applying click
reaction to doxycycline research.
5.1 Chemistry 5.1.1 Alkyne / Azide Derivatives and Reaction Optimization In order to investigate the click reaction, it is necessary to synthesize doxycycline
derivatives bearing either an alkyne or azide moiety. To accomplish that, I decided to
rely upon the acylation of 9-amino doxycycline (39) by reacting it with the adequate
anhydrides.
Concerning the synthesis of an alkyne derivative, I started from the commercially
available 5-hexynoic acid and synthesised a mixed anhydride with isobutyric acid
chloride (scheme 5.1.1a).
Scheme 5.1.1a : Synthesis of hex-5-ynoic isobutyric anhydride.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
55
The reaction of anhydride 50 with 9-amino doxycycline afforded 9-(hex-5-ynamido)-
doxycycline (51), as depicted in scheme 5.1.1b.
Scheme 5.1.1b : Synthesis of 9-hex-5-ynamido-doxycycline.
In analogy to the synthesis of the azido bearing derivative, I started from ethyl-5-
bromovalerate to yield the azido acid derivative 52a, as described in the literature.(7)
Then, reacting it with dicyclohexyl carbodiimide I obtained the symmetrical anhydride
52b, as in scheme 5.1.1c. The insertion of the azide was accomplished referring to a
microwave procedure reported by Rajender et al. (8)
Scheme 5.1.1c : Synthesis of 5-azidopentanoic anhydride.
The reaction of anhydride 52b with 9-amino doxycycline afforded the derivative 53, 9-
(5-azidopentanamido)-doxycycline, as depicted in scheme 5.1.1d.
Scheme 5.1.1d : Synthesis of 9-(5-azidopentanamido)-doxycycline.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
56
Stability Studies.
I decided to investigate the stability of the alkyne bearing doxycycline derivative 51 in
the two different systems usually adopted to pursue the click reaction, i.e. the
aqueous cycloaddition procedure (copper sulphate and sodium ascorbate in aqueous
media)(9) and the organic solvent procedure (copper iodide with DIPEA in organic
solvent).(10)
This investigation was necessary because many reports anticipated possible
problems in the reaction I was going to develop. First of all, we are dealing with very
sensitive molecules that do not tolarate a variety of physico-chemical stress (extreme
pH values, light, high temperatures, O2).(11)
Moreover, it is known from the literature that tetracyclines are rapidly degraded by
copper-base complexes,(12) that they form stable complex with metal ions (copper II
included), and that they exhibit poor solubility in different solvents. The complexation
of copper (II) ions and the solubility are crucial factors within our study, the first
because only copper (I) catalyze the click reaction, and the sequestration of copper
(II) by tetracycline could impede the catalysis; the second, because often solubility
was a limiting factor in the success of click reactions.(13)
As shown in table 1, in the aqueous systems (water plus alcohol), the derivative 51 is
not soluble. Another negative factor is the general tendency of degradation of the
molecule on the long time, especially in the copper (I) - DIPEA system. This tendency
is even more marked if the temperature is raised.
Solubility Stability r.t.
24h Stability 40°C
24h Stability 60°C
24h
H2O / MeOH CuSO4 NaAsc - + + + -
H2O / t-BuOH CuSO4 NaAsc - + + + -
H2O / DMF CuSO4 NaAsc + + + - + -
DMF DIPEA CuI + + - - -
Table 1 : Investigation of the stability of 51 in various “click” conditions.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
57
Reaction optimization
In order to optimize the reaction conditions of the triazole formation, the reaction
between compound 51 (9-(hex-5-ynamido)-doxycycline) with benzyl azide to give
compound 54 (9-(4-(1-benzyl-1H-1,2,3-triazol-4-yl) butanamido)-doxycycline) was
investigated. A series of experiments with variations in terms of solvent, catalyst, and
temperature was carried out.
Scheme 5.1.1e : Model reaction for the optimization of catalyzed cycloaddition.
Seven different procedures were investigated, all based on the aqueous
methodology, because the stability studies clearly indicated a better tolerance of the
doxycycline derivative to such systems. Table 2 gives an insight into the used
systems.
SOLVENT CATALYST REDUCING AGENT LIGAND
1 H2O / MeOH CuSO4 Sodium
Ascorbate
2 H2O / MeOH CuSO4 Sodium
Ascorbate Bathophenanthroline
disulfonic acid
3 H2O / MeOH CuSO4 TCEP
4 H2O / MeCN CuSO4 Sodium
Ascorbate
5 H2O / DMF CuSO4 Sodium
Ascorbate
6 H2O / t-BuOH CuSO4 Sodium
Ascorbate
7 H2O / MeCN [Cu(CH3CN)4][PF6] Table 2 : Click system used for reaction optimization.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
58
Each of this systems was studied with the introduction of three catalyst loadings
(10%, 30% and 1.1 equivalents, with reducing agent loading double as of catalyst),
and different reaction temperature were applied: no heating, 40°C, 60°C, and
microwave irradiation with 50 or 100 watts.
Not even one of these combinations gave a complete conversion of the reactant into
the product 54, and when the catalyst loading was 10% or 30% absolutely no
reaction occurred. Instead the progressive degradation of the doxycycline derivative
was confirmed.
A deeper insight into the investigated systems is necessary to reveal the rationality
behind the undertaken attempts.
Systems 1 and 6 represent typical click reaction conditions, i.e. CuSO4 / sodium
ascorbate catalyst system in a mixture of alcohol (MeOH or t-BuOH) and water.
Since all of the combinations were unsuccessful, at first other solvent systems were
investigated where the doxycycline derivative was soluble, namely acetonitrile / water
(system 4) and DMF / water (system 5).
Assured that the reaction’s failure was not due to solubility problems, other variables
had to be changed. System 3 investigated the use of an alternative reducing agent,
tris(carboxyethyl)phosphine hydrochloride (TCEP), used by different research groups
when applying click chemistry to sensitive molecules.(14,15,16)
The use as additive of bathophenanthroline disulfonic acid sodium salt in system 2,
followed the finding of Fokin and Finn that in 2004(17,18) reported the enhancement of
copper catalytic activity by different ligands. This protocol was successfully employed
by the Wang group, who chemoselectively functionalized the cage-like protein ferritin
and labelled it via CuAAC with a coumarin derivative.(19)
Lastly, in system 7 the stabile Cu(I) catalyst, tetrakis(acetonitrile)copper(I)
hexafluorophosphate (Cu(CN)4PF6 ) was used, which was applied for example in
carbanucleosides research by Agrofoglio et al.(20)
Many of the above mentioned alternatives were methodologies developed because
the classical click reaction conditions failed, and that usually because researcher
were dealing with complex chemical or biological structures.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
59
Regardless of the major risk of degradation of the doxycycline derivatives, the
organic solvent-based procedure had to be exploited. In fact, this procedure was
successfully applied where the aqueous cycloaddition failed, particularly in the field of
polymer science and biohybrid materials.
To investigate this alternative, we referred to the first publication on the CuAAC by
Meldal et al.(21) that used the organic solvent procedure with two equivalents of
copper iodide and a large excess of base (50 equivalents) to afford the triazole
derivatives.
Indeed, applying this method, a good conversion of hexynoyl derivative 51 into the
“clicked” derivative 54 could be observed, although slowly.
It seemed that the bivalent metal complexation capacity of tetracyclines was probably
responsible for the failure of the aqueous procedure; it is possible that the copper(II)
ions were sequestrated by chelation with doxycycline, and they could no more be
reduced by ascorbate catalyzing the cycloaddition. The catalyst loading of 1.1
equivalents was insufficient as well, probably because each tetracycline molecule
can complexate more than one copper ion, as reported in literature.(22-25)
In order to speed up the click reaction, we applied a procedure developed by
Kirshenbaum et al., who investigating the CuAAC in peptoid research taked
advantage of a large excess of copper iodide (13 equivalents) to catalyze the
cycloaddition reaction.(26) This procedure permitted the achievement of a total
conversion of the derivative 51 into 54 within one hour. This result was favourable
welcomed, since a short reaction time is fundamental to avoid side products, that
occur on longer periods.
Regioselectivity Investigations
The cycloaddition of azide with terminal acetylenes conducted thermally is non-
regiospecific and gives two possible isomers, the 1,4 (anti) and the 1,5 (syn) triazole.
Catalysis of the Huisgen 1,3 cycloaddition with different metals speeds up the
reaction and permits an high regioselectivity control. To achieve 1,4 disubstituted
triazoles copper catalysts are used, whilst using ruthenium catalyst only 1,5 products
are obtained.
In order to confirm the achievement of a 1,4 triazole, the derivative 54 was carefully
investigated using two-dimensional nuclear magnetic resonance spectroscopy. The
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
60
correlation of the nuclei confirms the position of the substituents, since a 1,5 isomer
would not show an interaction between the triazole proton and the methylene of
benzyl group (figure 5.1.1f).
Fig. 5.1.1f : HMBC study to confirm the obtainment of a 1,4 substituted triazole derivative.
5.1.2 Functional Groups Tolerance Studies To assume that the optimized reaction conditions were robust and reliable, the
reactivity of the alkyne derivative 51 and the azido derivative 53 were firstly
investigated by reacting them with different building blocks bearing diverse chemical
moieties.
Derivative 51 (alkyne bearing doxycycline) was reacted with four building blocks
bearing functionality such as aromatics, carboxylic acids, esters and azido acid ester.
As described previously, reaction with benzyl azide afforded derivative 54. Reaction
with the abovementioned ethyl 5-azidopentanoate, yielded compound 55 (ethyl 5- [4-
[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl] pentanoate); reaction with
methyl 2-azido-3-phenylpropanoate, an azido derivative of the methyl ester of
phenylalanine, gave the cycloadduct 56 (methyl 2- [4-[4-(doxycycline-9-ylamino)-4-
oxobutyl]-1H-1,2,3-triazol-1-yl]-3-phenylpropanoate); finally, reaction with an azido
derivative of the mini-PEG-3 linker afforded the respective derivative 57 (scheme
5.1.2a).
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
61
OH
NH
O
3
NN
N
OH
NH
ONN
NO
4
OH
NH
O
3
NN
N
OH O OH O
OH
O
NH2
CH3 OH
OH
N
NH
O
3
3
OH
NH
O
3
NN
NO
3
O
O
O
HO
O
51
54
55
56
57
a.
a. CuI (13eq), Ascorbic Acid (7eq)DIPEA (17eq), DMF, 40°C, 1h
Scheme 5.1.2a : Synthesis of derivatives 54, 55, 56 and 57 starting from the alkynoyl derivative.
Azido doxycycline derivative 53 was reacted with building blocks containing
functionalities such as esters, protected amine, and free amine. Reaction with hex-5-
ynoic acid afforded derivative 58 (4-(1-(5-(9-amino-doxycycline)-5-oxopentyl)-1H-
1,2,3-triazol-4-yl) butanoic acid); reaction with N-Boc protected propargylamine and
with propargylamine afforded respectively compounds 59 (tert-butyl (1-(5-(9-amino-
doxycycline)-5-oxopentyl)-1H-1,2,3-triazol-4-yl) methylcarbamate) and 60 (9-(5-(4-
(aminomethyl)-1H-1,2,3-triazol-1-yl) pentanamido)-doxycycline).
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
62
Scheme 5.1.2b : Functional groups tolerance studies on compound 53.
Compounds 55, 57 and 58 were also synthesized having in mind their strategical use
as linkers for coupling with amino acids and peptides.
5.1.3 Amino Acids Conjugates
The emergence of bacterial resistance on second-generation tetracyclines led to a
renewed interest in the synthesis of analogues that could circumvent existing
resistance mechanisms. This approach was thoroughly investigated by different
medicinal chemist groups arriving to the discovery and development of the
glycylcyclines.
As the name suggests, they are glycine derivatives of second generation molecules
such as minocycline, sancycline and doxycycline. The conjugation between the
tetracyclines core and the glycine subunit is done by an amide bond between the
aniline functional group in position 9 and the carboxylic group of the amino acid
derivative.
Figure 5.1.3a :Tthird generation tetracyclines. The blue dotted square shows the conjugated amino
acid functionality. For tigecycline structure see figure 1.2.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
63
The synthesis of clicked conjugates through its triazole linkage offers a rational
alternative to obtain similar analogues. 1,4 triazoles are in fact acknowledged amide
isosters, because of the similarity in rests distancing (3.9 Angstrom in peptide bond
versus 5.0 in 1,4 triazoles) and posses the same ability in forming H-bonds.
Changing from amide to triazoles in molecules where the peptide bond was a central
pharmacoforic unit was also successful, fully confirming this bioisosteric rule.(27)
Amino acids building block synthesis: N-terminus
Scheme 5.1.2c depicts the strategy used to build N-terminus modified amino acids.
The azide/alkyne functionalized amino acids were synthesized on 2-chloro-trityl resin
following an in-house developed procedure of Fmoc-based microwave solid-phase
peptide synthesis (3 equivalents of Fmoc-amino acid and DIPEA; 20% pyridine in
DMF for 10x10 seconds; PyBOP, HOBt and DIPEA for 15x30 seconds, were used for
the loading (a), deprotection (b) and coupling step (c), respectively). The coupling
reaction was done with hex-5-ynoic acid to obtain the alkyne functionalized amino
acid, and with 5-azidopentanoic acid to achieve the azide derivatized one. The
accomplishment of the coupling reaction was tested by Kaiser test at each step.
Cleavage was effected by reaction with TFA/DCM/TIS (10:85:5) for 30 min (d). The
building blocks were synthesized on the basis of glycine, alanine and phenylalanine.
The resulting modified amino acids were used without further purification for the click
reaction with doxycycline derivatives.
Scheme 5.1.3a : Synthesis of azide / alkyne amino acids building blocks.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
64
Clicked Conjugates
The conjugation between doxycycline and amino acids or peptides was particularly
challenging, because of their ability to coordinate copper ions through the nitrogen
and oxygen atoms of the amido functionality. The combination of doxycycline´s
complexation ability and peptides´ coordination capacity could be disadvantageous,
as already experienced with doxycycline when optimizing the click reaction
conditions. Indeed, the reactions of doxycycline derivatives with the amino acids
modified building blocks were clearly slower, and for some substrates no complete
conversion was observed even after seven hours reaction time. To solve this
inconvenience, I decided to carry out the reaction employing microwave irradiation,
that in recent times was adopted with great success to improve organic synthesis,
especially in the field of metal catalyzed reactions. (29)
Different research groups had to modify their procedure when applying the click
reaction for peptide or protein modification, and microwave irradiation was also
successfully applied, reducing reaction times down to 3 minutes.(30,31,32,33)
Irradiation of the same substrates with microwaves permitted to obtain a complete
conversion of the starting material into the derived amino acid conjugates within 5
minutes. To take full advantage of the microwave process, the reaction was pursued
in cycles of 30 seconds each, cooling the vials to zero degrees between the cycles.
This rapidity of the conversion into the triazole linked conjugates was eagerly
welcomed, considering the stability problems of the derivatives as reported in
paragraph 5.1.1.
Starting form alkyne derivative 51 I synthesized conjugates with the azido glycine
(67), azido alanine (68) and azido phenylalanine (69), as depicted in scheme 5.1.3b.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
65
Scheme 5.1.3b : Synthesis of amino acid conjugates starting from doxycycline alkyne derivative.
Reacting compound 53 (azido doxycycline derivative) with alkyne modified glycine,
alanine and phenylalanine I obtained derivatives 70, 71 and 72, respectively (scheme
5.1.3c).
Scheme 5.1.3c : Synthesis of amino acid conjugates starting from doxycycline azido derivative.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
66
Esterification of the acid functionality
Tetracyclines diffuse passively across the lipid bilayer of bacterial membranes. As
reported in paragraph 3.2, chemical modifications could annul this property, thus
rendering the derivatives inactive. The “clicked” amino acids conjugates are surely
more hydrophilic than the mother molecule, because of the presence of the triazole
group and the carboxylic acid functionality. In order to raise the lipophilicity of the
compounds, the free carboxylic acid functionality was treated with methanol/HCl to
obtain the respective methyl ester derivatives. This is a classical medicinal chemistry
methodology to increase the probability of passing cell membranes.(34)
The scheme 5.1.3d shows the compounds thus obtained.
OH
NH
O
3
NN
NO
NH
HO
O4
OH
NH
O
N
3
NNO
NH
HO
O
4R
R
OH
NH
O
3
NN
NO
NH
O
O4
R
OH
NH
O
N
3
NNO
NH
O
O
4R
MeOH / HCl
MeOH / HCl
(67) R = H(68) R = CH3(69) R = CH2Phe
(73) R = H(74) R = CH3(75) R = CH2Phe
(70) R = H(72) R = CH2Phe
(76) R = H(77) R = CH2Phe
Scheme 5.1.3d : Esterification of the carboxylic acid functionality of the clicked amino acid conjugates.
Amino acids building blocks synthesis: C-terminus
Thinking about the following application of the click methodology for the ligation with
peptides, I decided to synthesize analogues to the above described conjugates with
C-terminally modified amino acids. To obtain a high grade of homology, the
derivatization should maintain a linear approach. I thus started from commercially
available amino acids or dipeptides (three N-Boc protected and one N,N-
dimethylamino modified) and coupled them in liquid phase with 3-azido-prop-1-
ylamine. This linker was synthesized as described in the literature starting from 3-
bromopropylamine.(35)
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
67
The strategical approach is represented in scheme 5.1.3e.
Peptide bond formation was pursued in DMF, using HOAt and EDC as reagents and
triethylamine (TEA) as base. The products derived from the reaction are soluble in a
citric acid solution, allowing the isolation of the amino acid derivative through
extraction, avoiding a chromatographic purification. Cleavage of the protecting group
afforded the free amino derivatives. In contrast, the N,N-dimethylamino glycine
derivative was purified by flash chromatography and of course no deprotection step
followed.
Scheme 5.1.3e : Strategical approach for the C-terminus modification of amino acids building blocks.
Four building blocks were obtained following this procedure: N-(3-azidopropyl)-2-
(dimethylamino) acetamide (78), starting from N,N-dimethylglycine; (S)-2-amino-N-(3-
azidopropyl) propanamide (79), starting from Boc-alanine; (S)-2-amino-N-(3-
azidopropyl)-3-phenylpropanamide (80), starting from Boc-phenylalanine; and (S)-2-
amino-N-(2-(3-azidopropylamino)-2-oxoethyl) propanamide (81), starting from Boc-
alanine-glycine dipeptide (scheme 5.1.3f).
N
O
NH
O
NH
O
NH
O
OHN
N
HN
O
N3
H2N
HN
O
N3
H2N
HN
O
N3
NH
HN
O
N3
O
H2N
OH
OH
OH
OH
O
O
O
O
O
O
a.
(chromatography)
a, b
a, b
a, b
a. HOAtEDCTEADMF, 0°C --> r.t24h
b. TFA/DCMr.t. 1-3h
78
79
80
81
Scheme 5.1.3f : C-terminus modified building blocks.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
68
Clicked Conjugates
Following the developed procedure for the amino acid „click“ conjugates, starting
from 51 the C-terminus modified building blocks were successfully coupled. Scheme
5.1.3f shows the derivatives thus obtained. Conjugation with N,N-dimethylamino-
glycine afforded compound 82; conjugation with alanine and phenylalanine building
blocks gave compounds 83 and 84, respectively; conjugation with the modified
alanyl-glycine resulted in derivative 85.
OH
NH
O
3
NN
NHN
H2N
O3
OH
NH
ONN
NHN
N
O3
OH
NH
O
3
NN
NHN
NH
O
O
H2N
OH O OH O
OH
O
NH2
CH3 OH
OH
N
NH
O
3
51
a.
a. AA (2eq), CuI (13eq),Ascorbic Acid (7eq),DIPEA (17eq),DMF, MW 100W 10x30"
OH
NH
O
3
NN
NHN
H2N
O3
3
3
83
82
84
85
Scheme 5.1.3g : Synthesis of C-terminus modified clicked conjugates.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
69
5.1.4 Peptide Conjugates
The most revolutionary impact of the click reaction can be probably considered its
application in bioconjugation chemistry, representing an interdisciplinary field
between molecular biology and organic chemistry. Hereby, the employment of
reliable and selective reactions is fundamental, because the ligation between diverse
subunit must be not only chemically orthogonal, but also non-interacting (non-toxic)
with biological functionality while proceeding under physiological conditions.
Orthogonality means that the coupling partners of the reaction do not interact with
other functionality present in the reaction environment (e.g. vial or living organism).
The easy introduction of the azide and the alkyne functionalities, their inert properties
toward other moieties and thus their quite monogamous chemical reactivity, made
them ideal for application in bioconjugates research. As a result, numerous
biomolecules including DNA, peptides, proteins, oligosaccharides and
glycoconjugates have been modified via click chemistry and many of these new
molecular entities have proven extremely useful in the study of biological systems
(see references chapter 2, n. 15,16 and 17).
The application of click chemistry in bioconjugation was impressively refined by
Bertozzi,(36) who developed a copper free version of the click reaction, employing
cyclooctyne as dipolarophiles. This permitted an employment of this strategy not only
for in vitro studies, but also for biological investigations within intact cells and whole
organisms.
Our intention of developing doxycycline-peptides conjugates is driven by the interest
of providing the molecular biologist new molecular tools for a deeper investigation of
the tetracycline-TetR mediated transcription regulation.
The capacity of stem cells to develop into different types of specific cells with well-
defined functions in different organs is also linked to how the transcription is
regulated. Understanding more about the transcription process is therefore important
for the development of different therapeutic applications.(37)
The research was focused on conjugation with small peptides that act as co-
regulators. One of the two peptides, called VP1, is the minimal active sequence
derived from the herpes simplex transactivation protein VP16. This peptide is part of
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
70
the chimeric TetR protein used in the Tet-on and Tet-off expression systems (see
also paragraph 2.3).
Regarding the choice of a repressor peptide, a sequence containing the WRPW (Trp-
Arg-Pro-Trp) motif was synthesized. As proved by Caudy et al.,(38) this motif acts not
only as a direct transcriptional repressor, but is also capable of recruiting different co-
repressors via protein-protein interactions.
Strategy: N-Terminus modified Peptides
The synthesis of N-terminus modified peptides follows a procedure developed in our
research group by Dr. Einsiedel. Peptides are synthesized on solid-phase support
(rink-amide resin) using commercially available Fmoc-protected amino acids.
Coupling and deprotection cycles are carried out using microwave irradiation,
methodology that allows to speed up the process, contemporary increasing purities
and lowering racemization rates.
In analogy of the previously described amino acids building blocks, peptide of interest
were linearly built on the resin, and modified at the terminal amino acid with hex-5-
ynoic acid to obtain the alkyne functionalized peptide, and with 5-azidopentanoic acid
to achieve the azide derivatized one, respectively. The three aspartic acid residues
present in the sequence were protected with the acid labile tert-butyl ester.
In this way, six peptides were synthesized. Based on the VP1 sequence, derivatives
with and without the solubilizing linker mini-PEG-3 were made. The other two
peptides were based on the sequence SMWRPWRNG, with the azido or the alkyne
“click” group on the N-terminus.
After the completion of the synthesis, treatment with acid permitted the contemporary
cleavage of peptides from the resin and of the protecting groups from the amino
acids residues. The purity obtained by the methodology permitted to couple them to
the doxycycline derivatives without the necessity of a further purification.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
71
Click Conjugates
Using the developed methodology, a complete conversion into conjugates can be
observed under mild heating (50°C) within one hour. The presence of ascorbic acid is
crucial for the success, since analogous conditions without the presence of ascorbic
acid led to several peaks in the chromatogram of the LC-MS analysis, indicating the
possible formation of side reactions products. Scheme 5.1.4a shows the clicked
conjugates obtained between alkynoyl modified doxycycline 51 and azido
functionalized peptides: derivatives 86 and 87 are respectively the conjugates with
VP1 and with VP1 bearing the mini-Peg-3-linker; 88 is the cycloaddition product
between doxycycline and the inhibiting peptide containing the WRPW sequence.
Scheme 5.1.4a : Synthesis of peptide conjugates starting from alkynoyl doxycycline and azido peptides.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
72
Similarly, reaction of the azido derivative 53 with the alkynoyl modified peptides led to
analogous conjugates 89, 90 and 91, as depicted in scheme 5.1.4b.
Scheme 5.1.4b : Synthesis of peptide conjugates starting from azido doxycycline and alkynoyl peptides.
The successful development of both strategies was necessary because, as
previously reported (chapter 3, reference 18), it seems that the linkers play an
important role in such derivatives. Since the yields are comparable, it is possible to
use them indifferently, choosing the linker orientation that assures the best biological
properties.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
73
Strategy: C-terminus modified Peptides
The synthesis of click chemistry conjugates via their C-terminus is necessary to
elucidate which peptide orientation is more favourable for better
transactivation/transcription repression properties.
As the synthesis of linear analogues of the N-terminus modified peptides can not be
done using classical solid phase approach, so we investigated two alternative
strategies.
The strategies relied on the BAL (backbone amide linker) resin developed by Barany
and Albericio,(39) and on the “safety-catch” resin developed by Kenner and
Ellmann.(40)
The BAL resin presents an aldehyde group as anchoring functionality. A primary
amino linker containing a click moiety (azide or terminal alkyne) is incorporated via
reductive amination obtaining a secondary amine intermediate. This intermediate is
then acylated by the carboxyl carbon of the first amino acid, and classic solid phase
peptide synthesis procedure follows. Finally, the C-terminus modified peptide can be
cleaved from the resin with a mixture of trifluoroacetic acid and dichloromethane.
Scheme 5.1.4c : BAL strategy for C-terminus modified peptides. a) reductive amination b) acylation c)
removal of amine protecting group d) SPPS e) cleavage.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
74
The “safety-catch” resin is characterized by the presence of an alkanesulfonamide
rest. On this group, the peptide is built as usual; treatment with iodoacetonitrile then
provides an activated N-cyanomethyl derivative that can be cleaved with a variety of
nucleophiles to provide the C-terminal modified peptide.
Scheme 5.1.4d : „Safety-catch“ resin strategy. a) SPPS b) activation with iodomethane
Unfortunately, using these strategies it was not possible to afford peptides in
satisfactory yield and purity.
We therefore decided to adopt approaches that could be developed on the same
resins utilized for the N-terminus modified peptides.
The simplest strategy was the insertion in the sequence of a non natural amino acid,
bearing in the alpha carbon a moiety able to react via click chemistry, i.e. azide or
alkyne. We opted for the commercial available propargyglycine, obtaining a peptide
based on the VP1 sequence (P1).
Scheme 5.1.4e : Synthesis of the C-terminus modified peptide P1.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
75
The second strategy uses a N-alkylated peptide derivative. The amine residue
present in the Rink-Amide resin is firstly acylated with bromoacetic acid in presence
of diisopropylcarbodiimide (step a). The second step (b) involves nucleophilic
displacement of the bromine with 3-azido-propylamine, obtaining a secondary amine.
The peptide is built on this peptoid monomer and finally cleaved from the resin with
95% TFA in water, obtaining peptide P2.
Scheme 5.1.4f : Synthesis of the C-terminus modified peptide P2.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
76
Click Conjugates
With the same methodology used when conjugating the N-modified peptide
derivatives, the two peptides P1 and P2 were “clicked” together with doxycycline.
The peptide containing propargylglycine as first amino acid (P1) was coupled with the
azido derivative 53 and cojugate 92 was obtained.
The N-alkylated peptide (P2), presenting the azido functionality, was “clicked” with
alkynoyl doxycycline derivative 51 yielding the formation of the conjugate 93 (scheme
5.1.4c).
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
O
N
N N
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
ON
N N
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
O
N3
4
NH
H2N
O
O
N
H2N-Asp-Phe-Asp-Leu-Asp-Met-Leu-Gly4
3O
CONH2
3H2N-Asp-Phe-Asp-Leu-Asp-Met-Leu-Gly
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
O
3
53
51
92
93
P1 (1.5 eq)
P2 (1.5 eq)
CuI (13 eq) DIPEA (17 eq)ascobic acid (7 eq)DMF/CH3CN 50°C 1h
CuI (13 eq) DIPEA (17 eq)ascobic acid (7 eq)DMF/CH3CN 50°C 1h
Scheme 5.1.4e : Synthesis of C-terminus peptide conjugates.
The click reaction confirmed its reputation of ideal bioconjugation strategy, and its
applicability also for difficult structures such as doxycycline-peptide conjugates was
demonstrated. The orthogonality of the CuAAC permitted the reaction of fully
deprotected peptide fragments, avoiding solubility problems and saving the
deprotection step. The high purity obtained using our procedure for peptides
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
77
synthesis reduced the obtainment of pure conjugates to only one final HPLC
purification step.
The optimized procedure can be now exploited for the formation of conjugates with
diverse peptides, such as dimer or trimer of the VP1 sequence. These molecules will
hopefully give new insights into gene expression regulation research.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
78
5.2 Biological Investigations The amino acids conjugates were tested in the laboratories of microbiology
department by the group of Prof. Hillen, to investigate their ability of binding and
inducing different TetR mutants.
tTA [RLU, μg/protein]
rtTA-S3 [RLU, μg/protein]
rtTA-M2 [RLU, μg /protein]
rtTA-V16 [RLU, μg /protein]
rtTA-V10 [RLU, μg /protein]
None 12468,56 1,20 2,14 15,64 4,21 Dox 95,69 30,15 9017,76 1065,37 1847,42 56 117,72 963,18 614,88 642,30 346,81 75 57,16 215,77 139,88 1677,35 179,22 76 56,37 98,65 101,45 954,24 80,00 84 64,22 959,27 926,13 1526,80 1072,04 83 76,68 57,10 20,35 757,19 26,58 70 67,25 2,64 2,15 17,05 3,68 69 54,62 1358,33 6154,58 899,43 4372,64 72 95,51 2,38 4,15 41,38 5,12 71 159,46 2,15 3,03 6,30 4,57 68 86,41 4,37 2,87 60,04 2,44 74 82,23 17,41 11,36 433,23 16,16 77 62,71 51,12 4541,03 1116,03 4385,25 73 53,36 217,31 307,50 1484,28 1029,12 82 48,13 40,97 45,20 1342,16 67,85 85 68,43 10,46 3,37 305,97 15,44 67 85,35 2,45 2,61 8,24 6,07
Table 5.2 : Survey of the test results for doxycycline amino acids conjugates. Luciferase activity was
measured and standardized to Renilla luciferase activity. Induction ratios were estimated by standardized basal levels of luciferase activities [std. RLU, Doxy(–)] and standardized induced levels
of luciferase activities [std. RLU, Compound(+)].
Compounds were tested in vivo with cell lines expressing different TetR protein
mutants. tTA represents the TetR protein fused with the C-terminal portion of
transcription activator VP16. rtTA-S3, rtTA-M2, rtTA-V16 and rtTA-V10 are mutants
of the reverse phenotype of TetR, whose amino acids mutations are described in
references 41 and 42. These rtTA variants were chosen because of their higher
sensitivity toward doxycycline-like compounds. Among the four reverse TetR, the
rtTA-V16 seems particularly responsive to the conjugates.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
79
For all molecules, an in vivo inducer activity can be observed. This means that the
attachment of the peptide residues does not inhibit the diffusion into the cell. The
esterification developed on the carboxylic acids gave contrasting results: the esters
of glycine conjugates shows better inducing activity than the free carboxylic acid
derivatives; on the other hand, the analogue alanine conjugates did not reveal big
difference in potency; finally, phenylalanine derivatives gave contrasting results,
where compound 69 (carboxylic acid) shows bigger inducing potency than 75 (ester),
and derivative 72 (carboxylic acid) has worse activity than 77 (ester).
Generally speaking, the alanine conjugates show the worst inducing properties,
together with the alanine-glycine derivative 84. The glycine derivatives 67 and 70,
bearing a free carboxylic acid, show low inducing activity but their corresponding
methyl esters 73 and 76 induce much better at all the five proteins. It is not possible
to say if these different responses are due to better inducing properties or if the
diffusion increase caused by esterification has an important impact for glycine
derivatives, because of the marked hydrophily of the glycine rest.
Molecules bearing a phenylalanine rest are particularly active. As suggested by
Daam,(43) it is possible that this residue strongly interacts with the TetR protein in a
similar manner to that of Tip, a peptidic TetR inducer. Crystal structures of TetR in
complex with Tip clearly show the interaction of the rests W1-T2-W3-N4 with the
amino acids that constitute the tetracyclines binding pocket. Moreover, other eight
amino acids (Ala5 to Ser12) interact with residues toward the surface of TetR.
Phenylalanine 8 seems to play an important role, since it binds with six more amino
acids residues of the TetR protein (figure 5.2a).
Figure 5.2a : Interactions between Tip peptide (a) and tetracyclines (b) with TetR binding pocket.
Development of a Click-Chemistry Strategy for the Modification of Doxycycline.
80
If we now compare the structure of TIP peptide with the doxycycline-phenylalanine
conjugate 69, it is possible to notice the coinciding distance between the part of the
molecule that anchors to the binding pocket (red) and the phenylalanine rest
(coloured in blue). Moreover, the triazole moiety can mimic the peptide bond between
alanine 5 and tyrosine 6 (figure 5.2b).
Figure 5.2b : Similarities in the structures of compound 69 and Tip peptide. Red part shows the region
that binds in the tetracycline binding pocket. Blue region shows the phenylalanine rest.
On the basis of these speculations, these relationships are now being investigated in
the computer chemistry department of the university of Erlangen-Nürnberg, using
docking and scoring software. The synthesis of similar derivatives could corroborate
these hypotheses, eventually leading to twin drugs structures fusing together the
elements of tetracyclines with them of Tip peptides.
Moreover, important Tip residues can be inserted as linker between doxycycline-
peptide constructs, to afford a more efficient TetR binding and to properly distanciate
the inducer / silencer peptide from the repressor protein, in order to have a higher
probability of interaction with the transcription machinery’s proteins.
Summary
81
6. Summary Tetracyclines are widely used broad-spectrum bacteriostatic antibiotics that affect
both Gram-positive and Gram-negative bacteria, binding to the bacterial 30S
ribosomal subunit and inhibiting protein synthesis.
Recently the synthesis of tetracyclines analogues has gained renewed interest
because of the phenomenon of bacterial resistance, which is effected by tetracycline
efflux, ribosome protection and tetracycline modification.
The most important resistance mechanism in Gram-negative bacteria is the active
efflux of tetracyclines out of the cell by the membrane transport protein TetA, whose
expression is tightly regulated at the transcription level by the Tet-repressor (TetR)
protein. TetR binds with high specificity to its operator tetO and shows high affinity to
tetracyclines ensuring sensitive induction. These regulatory properties were exploited
by molecular biologist leading to tetracyclines-TetR based systems that allow selectiv
control of single genes expression in eukaryotes.
The aim of my work was the development of new semi-synthetic doxycycline
derivatives and could be subdivided into three topics, addressing different objectives:
I. Synthesis of novel 4-dedimethylamino-doxycycline derivatives, aiming at the
achievement of non antibiotic inducers for diverse TetR mutants.
II. Synthesis of doxycycline derivatives for the development of a SPR (Surface
Plasmon Resonance) biosensor for the analysis of tetracycline antibiotics
residues in foodstuffs.
III. Development of a click chemistry based approach for the structural modification
of doxycycline and its application for the synthesis of amino acids and peptides
conjugates.
Synthesis and Modification of 4-Dedimethylamino Doxycycline
Starting from doxycycline the 4-dedimethylamino derivative 3 can be afforded in a
two step synthesis including a methylation of the tertiary amino functionality and
subsequent reductive elimination.
Summary
82
Compound 3 was further modified by the introduction in the aromatic ring of amino
and halogen functionalities. Via nitration and following palladium catalysed
hydrogenation, 9-amino-4-DDMA-doxycycline (5) was synthesised. Using different
halogenation reagents, 9-bromo- (8), 9-iodo- and 7,9-diiodo-4-dedimethylamino
doxycycline (6 and 7) were afforded (scheme 6.1.1a).
Scheme 6.1.1a : Synthesis of 7- and 9- derivatives of 4-DDMA Doxycycline.
Starting from 9-amino-4-DDMA-doxycycline N-acylation gave different 9-amido-4-
DDMA-doxycycline, including 9-acetylamino- (12), 9-propionylamino- (13), 9-
benzoylamino- (14) and 9-pivaloylamino-4-DDMA-doxycycline (15).
Reductive alkylation with different aldehydes led to 9-dimethylamino-, 9-diethylamino-
and 9-dipropylamino-4-ddma-doxycyclines (16-18). Reaction with ketones led to the
respective derivatives N-isopropyl-, N-cyclopentyl-, N-cyclohexyl- and N-methyl,N-
isopropyl-amino-4-ddma-doxycycline (19-22) (Scheme 6.1.1b).
Summary
83
OH O OH O
OH
O
NH2
CH3 OH
OHH2N
reductiveamination
N-acylation
OH O OH O
OH
CH3 OH
OHCONH2N
H
O
R
R = CH3 (12), CH3CH2 (13)C6H5 (14), (CH3)3C (15)
OH O OH O
OH
CH3 OH
OHCONH2N
R1
R2
R1=R2 = CH3 (16)CH3CH2 (17)CH3CH2CH2 (18)
R1=H, R2 = isopropyl (19)R1=H, R2 = cyclopentyl (20)R1=H, R2 = cyclohexyl (21)R1=CH3, R2= isopropyl (22)
Scheme 6.1.1b : 9-Alkylamino- and 9-Amido-4-Dedimethylamino-Doxycycline.
Starting from 9-iodo- and 7,9-diiodo-4-DDMA-doxycycline, Sonogashira and Suzuki
palladium catalyzed cross-coupling reactions were investigated. Using Sonogashira
reaction various linear alkynyl (23, 25, 26) and a benzofuran (24) derivative were
obtained. Compound 24 is the product of a spontaneous 5-endo-dig cyclization from
the coupling reaction between 9-iodo-4-ddma-doxycycline and 5-hexynoic acid.
Analogous cyanation led to the nitrile derivative 28 (scheme 6.1.1c).
Scheme 6.1.1c : Synthesis of Sonogashira derivatives starting from 9-iodo-4-ddma doxycycline.
Summary
84
Starting from 7,9-diiodo derivative (7), reaction with 1 equivalent of phenylacetylene
led to selective substitution in position 9, obtaining compound 29, whilst using an
excess of reagent a disubstitution was obtained (compound 30, scheme 6.1.1d).
Scheme 6.1.1d : Synthesis of 7,9-disubstituded derivatives via Sonogashira reaction.
Derivatization of 9-iodo- and 7,9-diiodo-4-ddma doxycyclines via Suzuki-Miyaura
cross-coupling reaction afforded various phenyl bearing derivatives, such as 9-phenyl
(31), 9-para-carboxyphenyl (32) and 7,9-diphenyl (33), as illustrated in scheme
6.1.1e.
Scheme 6.1.1e : Derivatization of iodo doxycycline derivative via Suzuki-Miyaura reaction.
The biological investigations of 4-ddma derivatives gave interesting results with
bacterial strains possessing an increased membrane permeability, and opened new
questions about the importance of the dimethylamino group in position 4 for the
antibiotic activity of tetracyclines. Moreover, we found four derivatives with good
induction ability and selectivity toward the TetR i2 mutant.
Summary
85
Synthesis of modified doxycycline derivatives for SPR investigations
Surface Plasmon Resonance (SPR) is a modern methodology for studying biological
events such as affinity or association-dissociation kinetics. In collaboration with Prof.
Petz (University of Wuppertal), we decided to develop an SPR based system for the
rapid detection of tetracycline residues in foodstuffs. The central core of SPR
biosensors is formed by gold chips constituted by layers of different materials. The
upper layer presents carboxylic acid functionalities, where the molecular entities
under investigation can be bound using different coupling reactions.
Scope of the research was to attach a primary amino functionality to doxycycline with
different spacer lengths between the amino moiety and doxycycline core.
Starting from doxycycline, 9-amino-doxycycline (39) was synthesized via nitration
and subsequent reduction. Then, reaction with N-Boc-protected amino acid
anhydrides and eventual deprotection of the amino functionality with trifluoroacetic
acid led to the aminoacyl derivatives 43, 46 and 47. (scheme 6.1.1f).
Scheme 6.1.1f : Synthesis of amino bearing doxycycline derivatives for SPR studies.
The three derivatives were covalently bound to gold chips and the biosensor thus
obtained was used to study their binding properties to TetR protein. The applicability
of this biological system for competition assays with tetracyclines showed first
promising results that need however further optimizations.
Summary
86
Development of a click chemistry approach for the modification of doxycycline
Click chemistry is a concept in organic chemistry that count on few reliable reactions
to synthesize a variety of molecules using a modular approach. The copper catalyzed
triazole formation starting from azides and terminal alkynes is considered as the
perfect reaction embodying the click chemistry principles.
We decided to exploit this reaction for the chemical modification of doxycycline and
then applied it for the synthesis of conjugates with amino acids and peptides.
9-Amino-doxycycline (39) was N-acylated using appropriate anhydrides to obtain the
alkyne or azido bearing compounds 51 and 53, respectively.
Scheme 6.1.1g : Synthesis of 9-(5-azidopentanoyl)amino and 9-hex-5-ynamido doxycyclines.
In order to study the tolerance toward different chemical functionalities, derivatives 51
and 53 were reacted firstly with different building block, as aromatics (54), carboxylic
acid esters (55), azido acid esters (56), carboxylic acids (57 and 58), N-Boc
protected amines (59) and primary amines (60) (scheme 6.1.1h).
Summary
87
OH
NH
O
3
NN
NR
O4
OH O OH O
OH
O
NH2
CH3 OH
OH
N
NH
O
3
O
3
O
O
O
HO
O
51
54
55
56
57
O OH O
OH
O
NH2
CH3 OH
OH
N
OH O OH O
OH
O
NH2
CH3 OH
OH
N
NH
O
N3
4
58 59 60
53
OH
NH
O
N4
NN
R O OH O
OH
O
NH2
CH3 OH
OH
N
H2NHN
O
O
O
HO3
R N3R
CuI CuI
Scheme 6.1.1h : Reactivity studies for azido and alkyne derived doxycyclines.
Doxycycline amino acid conjugates
The synthesis of doxycycline amino acids conjugates via click chemistry can afford
chemical analogues of third generation tetracyclines, via a bioisosteric replacement
of an amide moiety by a triazole.
Glycine, alanine and phenylalanine were N-terminally modified on solid phase, by
coupling them with hexynoic acid or with 5-azido pentanoic acid. After acidic
cleavage from the resin the six functionalized derivatives 61-66 could be obtained
(scheme 6.1.1i).
Summary
88
Scheme 6.1.1i : Synthesis of N-terminus modified amino acids.
The reaction of the azido functionalized compounds 64-66 with the alkynoyl
doxycycline afforded compounds 67, 68 and 69. Click reaction of alkyne building
blocks 61-63 with azido bearing doxycycline yielded conjugates 70, 71 and 72.
The developed procedure utilizes copper iodide as metal catalyst source and the
employment of microwave irradiation cycles to accelerate the cycloaddition.
In a second step, the carboxylic acid functionalities of the conjugates were
esterificated with methyl alcohol to improve their lipophilicity, obtaining derivatives
73-77 (scheme 6.1.1j).
Summary
89
Scheme 6.1.1j : Synthesis of N-terminus modified amino acids conjugates.
Three amino acids and a dipeptide were modified at their C-terminus by amide bond
formation with 3-azidopropylamine in liquid phase. Thus, compound 78 was obtained
from N,N-dimethyl-glycine. Starting from N-Boc protected alanine, phenylalanine and
the dipeptide Boc-Ala-Gly-OH, compounds 79, 80 and 81 were afforded after
coupling with the azido linker and subsequent Boc deprotection (scheme 6.1.1k).
Scheme 6.1.1k : Synthesis of C-terminus modified amino acids building blocks.
Summary
90
Reaction of the C-terminally modified amino acids building blocks 78-81 with alkynoyl
doxycycline yielded the conjugates 82-85, utilizing the “click” methodology developed
for N-terminus modified amino acids conjugates (scheme 6.1.1l).
Scheme 6.1.1l : Synthesis of click conjugates between doxycycline and C-terminus modified amino
acids.
The amino acids conjugates were tested for their binding and inducing properties
towards different TetR systems. All molecules proved to be active in vivo, and some
of them show an increased inducing activity than doxycycline, in particular the
phenylalanine bearing conjugates (compounds 56, 69, 75 and 77).
Doxycycline-Peptide Conjugates
The application of click chemistry in peptide conjugation has the great advantage of a
complete orthogonality, offering the possibility to pursue the conjugation step with full
deprotected peptides.
For the synthesis of N-terminus linked peptides, both hexynoic acid and
azidopentanoic acid were coupled to the transactivating peptide VP1 and a peptide
containing the transcription inhibitory domain WRPW. The peptides were build via
SPPS using an Fmoc strategy.
The linkage strategy was exploited in both directions, starting from alkynoyl
doxycycline and azido modified peptides, or vice-versa starting from azide bearing
doxycycline conjugated with alkyne modified peptides.
Summary
91
HN-Asp-Phe-Asp-Leu-Asp-Met-Leu-GlyO
n
O
NH2
ONH2
O
3
OHOOHO
HO
CH3OH
OHH2NOC
N
NH
O
R
n
OH
NH
O
nOOHO
HO
CH3OH
OHH2NOC
N
HN-Asp-Phe-Asp-Leu-Asp-Met-Leu-GlyO
n
O
NH
OH
NH
O
nOOHO
HO
CH3OH
OHH2NOC
N
OH
NH
O
nOOHO
HO
CH3OH
OHH2NOC
N
HN-Ser-Met-Trp-Arg-Pro-Trp-Arg-Asn-GlyO
n
O
NH2
n = 3,4 R = N3,
86, 89
87, 90
88, 91
NN
N
NN
N
NN
N
Scheme 6.1.1m : Synthesis of doxycycline-peptide conjugates via the N-terminus.
For the attachment of the peptide VP1 via their C-terminus, two strategies were
adopted, incorporating in a case propargylglycine in the sequence, and in the other
an N-alkylated glycine scaffold bearing an azide group.
The click reaction with the modified doxycycline derivatives afforded the peptide
conjugates 92 and 93.
Summary
92
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
O
N
N N
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
ON
N N
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
O
N3
4
NH
H2N
O
O
N
H2N-Asp-Phe-Asp-Leu-Asp-Met-Leu-Gly4
3O
CONH2
3H2N-Asp-Phe-Asp-Leu-Asp-Met-Leu-Gly
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
O
3
53
92
51
93
Scheme 6.1.1n : Synthesis of doxycycline-peptide conjugates via the C-terminus.
The doxycycline-peptide conjugates thus obtained are under investigation for their
cell permeability and TetR inducing properties, and the results will be used for the
rational development of a novel ligand-based TetR system for eukaryotic gene
regulation.
Zusammenfassung
93
7. Zusammenfassung Tetrazykline sind weit verbreitete, bakteriostatisch wirkende Breitspektrum-
Antibiotika, die durch Bindung an die 30S Untereinheit des bakteriellen Ribosoms
und die dadurch ausgelöste Hemmung der Proteinbiosynthese sowohl auf
grampositive wie auch auf gramnegative Bakterien anwendbar sind.
Das Interesse an dieser Verbindungsklasse ist durch das Phänomen der
Bakterienresistenz, welche durch aktiven Tetracyclinefflux, ribosomale
Schutzproteine und enzymatische Tetracyclinmodifikation ausgelöst wird, neu
entflammt.
Der wichtigste Resistenzmechanismus in gramnegativen Bakterien ist der, durch das
Membran-Transportprotein TetA vermittelte aktive Ausstrom der Tetrazykline aus der
lebenden Zelle. Die Expression von TetA wird durch den Transkriptionsgrad des
TetR - Proteins (Tet-Repressor) bestimmt. TetR bindet mit hoher Spezifität an
seinen Operator tetO, zeigt eine hohe Affinität zu Tetracyclinen und ermöglicht somit
eine sehr empfindliche Induktion. Molekularbiologen machten sich diese regulierende
Eigenschaft zu Nutze und entwickelten auf Tetracyclin-TetR basierende Systeme,
welche eine selektive Kontrolle zur Expression einzelner Gene in eurokaryotischen
Zellen erlauben.
Das Ziel meiner Forschung war die Entdeckung neuer semi-synthethischer
Doxycyclin-Derivate und kann in drei Themen unterteilt werden, die sich mit den
jeweiligen Zielsetzungen befassen.
I. Synthese neuer 4-Dedimethylaminodoxycyclin - Derivate mit dem Ziel nicht
antibiotisch wirksamer Derivate zur Induktion von TetR Protein Mutanten.
II. Synthese modifizierter Doxycyclin – Verbindungen zur Entwicklung einer SPR –
Methode (Surface Plasmon Resonance) für die Detektion von Tetrazyklin-Antibiotika
in Lebensmitteln.
III. Entwicklung eines Click-Chemie basierten Ansatzes zur Derivatisierung von
Doxycyclinen zur Synthese von Aminosäure- und Peptidkonjugaten.
Zusammenfassung
94
Synthese und Modifikation von 4 Dimethyamino Doxycyclin
Ausgehend von Doxycyclin läßt sich das 4- Dedimethylamino Derivat 3 in einer zwei–
stufigen Synthese darstellen, wobei als erster Schritt eine Methylierung der tertiären
Aminofunktion durchgeführt wird, welche schließlich reduktiv eliminiert wird.
Derivat 3 wurde weitermodifiziert durch die Einführung einer Aminogruppe und eines
Halogens in den aromatischen Ring. Durch Nitrierung und anschließende Pd-
katalysierte Hydrierung konnte 9-Amino-4-ddma-doxycyclin (5) synthetisiert werden.
Der Einsatz verschiedener Halogenierungsreagenzien lieferte die Derivate 9-Bromo-
(8), 9 Iodo- und 7,9-Diiodo-4-ddma-doxycyclin (6 und 7) (Schema 7.1.1a).
Schema 7.1.1a : Synthese der 7- and 9- Derivative von 4-DDMA Doxycyclin.
Die N- Acylierung von 9-Amino-4-DDMA-doxycyclin ergab eine Reihe verschiedener
9-Amido-4-DDMA-doxycyclin-Derivate, einschließlich 9-Acetamido (14), 9-
Propionylamdio (13), 9-Benzoylamido (14) und 9-Pivaloylamido-4-DDMA-doxycylin
(15). Die reduktive Alkylierung mit verschiedenen Aldehyden führte zu 9-
Dimethylamino- , 9 – Diethylamino- und 9-Dipropylamino-4-DDMA-doxycyclinen (16-
18). Die Reaktion mit Ketonen brachte die entsprechenden Derivate N-Isopropyl-, N-
Cyclopentyl-, N-Cyclohexyl- und N-Methyl,N-isopropylamino-4-ddma-doxycyclin (19-
22) (Schema 7.1.1b).
Zusammenfassung
95
Schema 7.1.1b : 9-Alkylamino- and 9-Amido-4-Dedimethylamino-Doxycyclin.
Ausgehend von 9-Iodo and 7,9-Diiodo-4-DDMA-doxycyclin wurde die
Durchführbarkeit von Palladium katalysierten Sonogashira- und Suzuki-artigen
Kreuzkupplungsreaktionen untersucht. Die Sonogashirareaktion führte zu
verschiedenen linearen Alkinyl- (23, 25, 26) und einem Benzofuranderivat (24).
Verbindung 24 ist das Produkt einer spontanen 4-endo-dig Cyclisierung aus der
Kupplungreaktion von 9-Iod-4-ddma-doxycyclin mit 5-Hexinsäure. Die Analoge
Einführung eines Nitrils ergab das Nitrilderivat 28 (Schema 7.1.1c).
Schema 7.1.1c : Synthese von Sonogashira-Derivatien von 9-Iod-4-ddma-doxycyclin.
Zusammenfassung
96
Durch die Reaktion mit einem Äquivalent Phenylacetylen konnte beim 7,9-Diiod-
derivat eine selective Substitution in Position 9 erreicht warden, welche die
Verbindung 29 liefert. Im Gegensatz dazu konnte mit einem Überschuß an Reagenz
ein Disubstution erreicht werden (30) (Schema 7.1.1d).
Scheme 7.1.1d : Synthese von 7,9-disubstituierten Derivaten durch die Sonogashira Reaction.
Die Derivatisierung von 9-Iodo- und 7,9-Diiodo-4-ddma-doxycyclinen mittels Suzuki-
Miyaura Kreuzkupplung brachte zahlreiche Phenyl substuierte Derivate, wie 9-
phenyl- (31), 9-para-carboxyphenyl- (32) und 7,9-diphenyl-4-ddma-doxycyclin (33)
hervor, wie das Schema 7.1.1e zeigt.
Scheme 7.1.1e : Derivatization of iodo doxycycline derivative via Suzuki-Miyaura reaction.
Die biologischen Untersuchungen der 4-ddma Derivate mit Bakterienstämmen, die
eine erhöhte Membranpermeabilität besitzen, brachten interessante Ergebnisse,
warfen aber auch neue Fragen über die Bedeutung der Dimethylamino-Gruppe in
Position 4 für die antibiotische Aktivität auf. Desweiteren fanden wir vier Derivate mit
guter Induktionsleistung und Selektivität gegenüber der TetR i2 Mutante.
Zusammenfassung
97
Synthese von modifizierten Doxycyclinderivaten von SPR Untersuchungen
„Surface plasmon resonance“ (SPR) ist eine moderne Methode um biologische
Vorgänge wie Affinität sowie Assoziations- und Dissoziationskinetiken zu
untersuchen. In Zusammenarbeit mit Prof. Petz (Universität Wuppertal) entschieden
wir uns, ein auf dem Prinzip der SPR basierendes System zur schnellen Detektion
von Tetracyclinrückständen in Lebensmitteln zu entwickeln. Den zentralen Kern der
SPR Biosensoren bilden Goldchips, die aus verschiedenen Materialschichten
bestehen. Die oberste Schicht präsentiert Carbonsäurefunktionalitäten, woran die zu
untersuchenden, molekularen Funktionseinheiten unter Zuhilfenahme
unterschiedlicher Kupplungsreagenzien gebunden werden können.
Ziel der Untersuchung war die Funktionalisierung des Doxycyclins mit einer primären
Aminogruppe in verschiedenen, spacerabhänigen Abständen zum Doxycyclin-Kern.
Die Modifizierung von Doxycyclin führte durch eine Nitrierung und anschließende
Reduktion zum 9-Aminodoxycyclin 39. Die Weiterreaktion mit N-Boc geschützten
Aminosäureanhydriden und einer eventuellen Entschützung der Aminofunktion mit
Trifluoressigsäure ergab die Verbindungen 43, 46 und 47 (Schema 7.1.1f).
Schema 7.1.1f : Synthese von Amino substituierten Doxycyclin Derivaten für SPR Untersuchungen
Zusammenfassung
98
Entwicklung einer Click-Chemie Methode zur Modifikation von Doxycyclin
In der organischen Chemie ist die so genannte Click – Chemie ein Konzept, das
einige zuverlässige Reaktionen zu Nutze macht, um eine Vielzahl an Molekülen in
Form eines baukastenartigen Ansatzes herzustellen. Die Kupfer - katalysierte
Triazolbildung ausgehend von Aziden und terminalen Alkinen wird als die perfekte
Click – Reaktion betrachtet, weil sie alle Prinzipien der Click Chemie enthält.
Wir entschieden uns dafür, uns dieser Reaktion bei der chemischen Modifikation von
Doxycyclin zu bedienen und verwendeten sie für die Synthese von Konjugaten mit
Aminosäuren bzw. Peptiden.
9 Amino-doxycyclin (39) wurde mit entsprechenden Anhydriden N-acyliert, um die mit
Alkin funktionalisierte Verbindung (51) sowie das Azid tragende Derivat (53) zu
erhalten.
Schema 7.1.1g : Synthese von 9-(5-Azidopentanoyl)amino und 9-hex-5-inamido doxycyclinen.
Um die Verträglichkeit gegenüber verschiedenen chemischen Funktionalitäten zu
untersuchen, wurden die Derivate 51 und 53 zuerst mit verschiedenen Bausteinen
umgesetzt, wie Aromaten (54), Carbonsäureestern (55), Azidocarbonsäureestern
(56), Carbonsäuren (57 und 58) und N-Boc-geschützten Aminen (59) (Schema
7.1.1h).
Zusammenfassung
99
OH
NH
O
3
NN
NR
O4
OH O OH O
OH
O
NH2
CH3 OH
OH
N
NH
O
3
O
3
O
O
O
HO
O
51
54
55
56
57
O OH O
OH
O
NH2
CH3 OH
OH
N
OH O OH O
OH
O
NH2
CH3 OH
OH
N
NH
O
N3
4
58 59 60
53
OH
NH
O
N4
NN
R O OH O
OH
O
NH2
CH3 OH
OH
N
H2NHN
O
O
O
HO3
R N3R
CuI CuI
Schema 7.1.1h : Reaktivitätsuntersuchungen für Azido and Alkyne tragende Doxycyclinderivate.
Doxycyclin – Aminosäure Konjugate
Die Synthese von Doxycyclin – Aminosäure Konjugaten mittels Click – Chemie kann
chemische Analoga einer dritten Generation von Tetracyclinen hervorbringen. Die
Amidfunktion wurde bioisoster durch ein Triazol ersetzt.
Glycin, Alanin und Phenylalanin wurden mittels Festphasensynthese, durch
Kupplung mit Hexin- bzw. 5-Azido-pentansäure an ihren N-Termini modifiziert. Nach
saurer Abspaltung vom Harz konnten sechs funktionalisierte Derivate 61-66 isoliert
werden (Schema 7.1.1i).
Zusammenfassung
100
Schema 7.1.1i : Synthese von N-Terminus modifizierten Aminosäuren.
Die Reaktion der Azid - funktionalisierten Aminosäuren 64-66 mit Alkinoyl -
doxycyclin ergab die Aminosäurekonjugate 67, 68 und 69. Die anschließende Click
Reaktion von Alkinbausteinen mit Azid tragendem Doxycyclin führte zu den Derivaten
70, 71 und 72. Die dafür entwickelte Vorgehensweise benutzt Kupferiodid als
Metallkatalysator und macht sich die Mikrowellenstrahlung zur Beschleunigung der
Cycloaddition zu Nutze. Um die Lipophilie zu erhöhen, wurden in einem zweiten
Schritt die Carbonsäurefunktionen der Konjugate zu den entsprechenden
Methylestern umgesetzt, wobei die Derivate 73 – 77 isoliert wurden (Schema 7.1.1j).
Zusammenfassung
101
Schema 7.1.1j : Synthese von N-Terminus modifizierten Aminosäurekonjugaten.
Drei Aminosäuren und ein Dipeptid wurden an ihren C-Termini mit 3 – Azidopropyl-1-
amin in das entsprechende Amid überführt. Dadurch wurde N,N-Dimethylglycin zu 78 umgesetzt. Ausgehend von N - Boc – geschütztem Alanin, Phenylalanin und dem
Dipeptid Boc-Ala-Gly-OH wurden die Verbindungen 79, 80 und 81 durch Kupplung
mit dem Azidlinker und anschließender Boc - Entschützung synthetisiert (Schema
7.1.1k).
Schema 7.1.1k : Synthese von C-Terminus modifizierten Aminosäurebausteinen.
Zusammenfassung
102
Die Reaktion dieser C- terminal modifizierten Derivate (78 - 81) mit Alkinoyl -
doxycyclin lieferte die Verbindungen 82 – 85, wobei die eigens für N-terminal
modifizierte Aminosäurekonjugate entwickelte Click Methode zum Einsatz kam
(Schema 7.1.1l).
Schema 7.1.1l: Synthese von Click Konjugaten von Doxycyclin and C-Terminus modifizierten
Aminosäuren.
Die Aminosäurekonjugate wurden auf ihr Bindungs- und Induktionsverhalten
bezüglich verschiedener TetR - Systeme getestet. Alle Moleküle sind in vivo aktiv,
einige - besonders Phenylalanin tragende Konjugate 56, 69, 75 und 77 - zeigen eine
höhere induzierende Aktivität als Doxycyclin.
Doxycyclin-Peptid Konjugate
Die Anwendung der Click Chemie für die Synthese von Peptidkonjugaten bringt den
großen Vorteil umfassender Orthogonalität mit sich, wodurch der eigentliche
Konjugationsschritt mit vollkommen entschützten Peptiden durchgeführt werden
kann.
Für die Synthese N-terminal verlinker Peptide wurden sowohl Hexinsäure als auch
Azidopentansäure an das transaktivierende Peptid VP1 und an ein, die
transkriptionshemmende Domäne WRPW enthaltendes Peptid gekuppelt. Die
Peptide wurden mittels SPPS synthetisiert, wobei mit einer Fmoc Strategie gearbeitet
wurde.
Die Verknüpfungsstrategie wurde in beiden Richtungen ausgeführt, ausgehend von
Alkinoyldoxycyclin und azid modifizierten Peptiden, oder vice versa ausgehend vom
Azid substituierten Doxycyclin, welches mit Alkin modifizierten Peptiden gekoppelt
wurde.
Zusammenfassung
103
HN-Asp-Phe-Asp-Leu-Asp-Met-Leu-GlyO
n
O
NH2
ONH2
O
3
OHOOHO
HO
CH3OH
OHH2NOC
N
NH
O
R
n
OH
NH
O
nOOHO
HO
CH3OH
OHH2NOC
N
HN-Asp-Phe-Asp-Leu-Asp-Met-Leu-GlyO
n
O
NH
OH
NH
O
nOOHO
HO
CH3OH
OHH2NOC
N
OH
NH
O
nOOHO
HO
CH3OH
OHH2NOC
N
HN-Ser-Met-Trp-Arg-Pro-Trp-Arg-Asn-GlyO
n
O
NH2
n = 3,4 R = N3,
86, 89
87, 90
88, 91
NN
N
NN
N
NN
N
Scheme 7.1.1m : Synthese der Doxycyclin-Peptid Konjugate über N-terminale Verknüpfung.
Für die Verknüpfung des Pepdides VP1 über dessen C-Terminus wurden zwei
Strategien angewandt. Im einen Fall beinhaltete die Sequenz Propargylglycin, im
anderen Fall ein N-alkyliertes Glycin mit Azid Gruppe.
Die Click Reaktion mit modifizierten Doxycyclin Derivaten führte zu Verbindung 92 und 93.
Zusammenfassung
104
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
O
N
N N
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
ON
N N
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
O
N3
4
NH
H2N
O
O
N
H2N-Asp-Phe-Asp-Leu-Asp-Met-Leu-Gly4
3O
CONH2
3H2N-Asp-Phe-Asp-Leu-Asp-Met-Leu-Gly
OH O OH O
OH
CH3 OH
OHCONH2
N
NH
O
3
53
92
51
93
Scheme 7.1.1n : Synthese der Doxycyclin-Peptid Konjugate über C-terminale Verknüpfung.
Die dadurch erhaltenen Doxycyclin-Peptid Konjugate werden gerade im Hinblick auf
Zellpermeabilität und TetR-induzierende Eigenschaften untersucht und die
Ergebnisse werden für die rationale Entwicklung eines neuartigen Liganden-
basierten TetR Systems für die Genregulation in eukaryotischen Zellen Anwendung
finden.
Experimental Part
105
8. Experimental Part Materials and Methods Doxycycline monohydrate was acquired from Heumann Pharma.
All chemicals and solvents were purchased at their purest grades from ACROS, FLUKA, ALDRICH
and NOVABIOCHEM and used without further purification.
2-Chlorotrityl resin was purchased from IRIS Biotech.
IR Spectra were registered on Jasco FT/IR 410 instrument, using a film of substance on a NaCl.
TLC analyses were performed on Merck 60 F254 aluminium sheets and analysed by UV light
(254nm), by iodine vapour or by ninhydrin.
Flash chromatographies were made using Silica Gel 60 (40-63 um) as stationary phase.
HPLC-MS analyses were conducted in an Agilent Binary Gradient System in combination with
ChemStation Software (MeOH 0.1% HCOOH / H2O 0.1% HCOOH) and UV detection at 254 or 220
nm. The column was a Zorbax SV-C18 (4.6 mm IDX250 mm, 5 um) with a flow rate of 0.5 mL/min.
Mass detection was pointed out with a Brucker Esquire 2000 Ion-trap mass spectrometer using APCI
or ESI ionization source. 1H and 13C – NMR spectra were recorded in solution with TMS as internal standard using Brucker
Avance 360 (360MHz) or Brucker Avance 600 (600MHz) FT-NMR-Spectrometer.
HR-EIMS spectra were recorded on a Jeol GCmate II spectrometer.
MPLC separations were performed on a Büchi Chromatography System (binary pump B-688, gradient
former B-687 and glass columns B-685) with UV detection at 254 nm using Europrep 60-30 C18
(Eurochrom®, Knauer) RP silica gel and CH3CN / 0.1 % aq. TFA as a solvent system. In all cases
HPLC grade solvents were used.
Preparative HPLC was performed on an Agilent 1100 system using RP-18 colums (Agilent Zorbax
300SB, 7mm or CS-Chromatographie Eurospher C-18, 7mm) and CH3CN / 0.1% aq. TFA or MeOH /
0,1% aq. TFA as solvent systems.
Analytical HPLC analyses were performed on an Agilent 1100 system using Zorbax Eclipse XDB-C8
84.6 mm x 150 mm, 5 um) and CH3CN / 0.1% aq. TFA / 0,1% aq. TFA (0-3 min. 10%; 3-25 min.
gradient 10 100%; 25-28 min. 100%; 28-30 min. 100 10%) as solvent systems with a flow rate of
0.5 mL/min.
Experimental Part
106
Nomenclature of the substances
The compounds are named and numbered after the conventional nomenclature used for tetracyclines,
based upon their naphtacene ring system, as depicted in figure 8.1.
Fig 8.1: Doxycycline and conventional numbering of the same.
Doxycycline is the trivial name for the corresponding systematic IUPAC name (2-(amino-hydroxy-
methylidene)-4-dimethylamino-5,10,11,12a-tetrahydroxy-6-methyl-4a,5,5a,6-tetrahydro-4H-tetracene-
1,3,12-trione.
The compounds derived from click reaction with amino acids building blocks were named assuming 9-
amino doxycycline as a substituent, when determined by priorities. In every case triazole molecules
names were generatad by Struct=Name Pro 11.0 software developed by CambridgeSoft and
uncorrected.
The assignment of protons and carbons signals in the interpretation of NMR spectra followed the
conventional tetracycline position numbering.
Experimental Part
107
Doxycycline methyliodide (2)
Doxycycline methiodid was prepared starting from 10.42 g (22.58 mmol) of doxycycline monohydrate
as described in the literature (methylation by methyliodide in THF for 4 days at r.t.)
Characterization Yield : 13.13 g (99.2 %) as yellow solid Analytical Data : C23H27N2O8I (MW = 585.46) APCI-MS m/z m/z = 461.0 [M – I- ] HPLC : tr = 13.4 min. purity > 99 % (254nm) IR (KBr) : 3540-3100, 2973, 2876, 1651, 1615, 1583 cm-1 1H NMR (360 MHz, Pyr d5) :
δ (ppm) = 1.72 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.65 (s, 1H, H-5a), 2.80 (dq, 1H, J = 12.7,7.2 Hz, H-6),
2.91 (dd, 1H, J = 12.1, 8.3 Hz, H-5a), 3.86 (s, 9H, N+(CH3)3 ), 4.27 (dd, 1H, J = 10.8, 8.3 Hz, H4), 5.71
(s, 1H, OH-5), 6.82 (d, 1H, J = 7.7 Hz, H-9), 6.99 (d, 1H, J = 8.4 Hz, H-7), 7.4 (t, 1H, J = 7.9 Hz, H-8),
9.95 (br s, 2H, NH2), 10.10 (s, 1H, OH-10), 10.31 (s, 1H, OH-3).
4-Dedimethylamino doxycycline (CMT-8) (3)
5 g (8.54 mmol) of derivative 1 were dissolved in 150 mL of 50% acetic acid and stirred at r.t.. 3 grams
of Zn dust were added and after 20 minutes the suspension was filtered through Celite. To the filtrate
were added 500 mL of water containing 5 mL of concentrated HCl. The precipitate thus formed was
stirred in an ice bath for 1h, filtered and dried o.n. at the oil pump.
Characterization Yield : 2.493 g (72%) as light yellow solid
Experimental Part
108
Analytical Data : C20H19NO8 (MW = 401,38) APCI-MS m/z = 402.0 [M+1]+ HPLC : tr = 19.4 min. purity > 99 % (254nm) HR-EIMS calculated : m/z = 410.1110 found : m/z = 410.1110 IR (film) : 3600-3100, 2974, 2876, 1649, 1613, 1577 cm-1 1H NMR (360 MHz, Pyr d5) :
δ (ppm) = 1.74 (d, 3H, J = 6.6 Hz, CH3 at C-6), 2.74 (dd, 1H, J = 12.5, 7.7 Hz, H-5a), 2.83 (dq, 1H, J =
12.8, 6.2 Hz, H-6), 2.93 (ddd, 1H, J = 10.9, 5.2, 2.5 Hz, H-4a), 3.57 (dd, 1H, J = 18.3, 2.4 Hz, H-4
alpha ), 3.66 (dd, 1H, J = 18.3, 5.2 Hz, H-4 beta), 4.26 (dd, 1H, J = 10.9, 7.7 Hz, H-5), 6.89 (d, 1H, J =
7.7 Hz, H-9), 7.00 (d, 1H, J = 8.4 Hz, H-7), 7.43 (t, 1H, J = 8.1 Hz, H-8), 9.72 (br s, 1H, NH2), 10.10 (br
s, 1H, NH2), 12.12 (s, 1H, OH-3).
13C NMR (90 MHz, Pyr d5) :
δ (ppm) = 15.02 (CH3), 29.66 (C-4), 37.94 (C-6), 42.94 (C-4a), 45.65 (C-5a), 67.97 (C-5), 74.25
(C-12a), 98.36 (C-2), 106.02 (C-11a), 115.06 (C-7), 115.11 (C-9), 160.70 (C-10), 172.70 (C-12),
174.03 (CONH2), 192.64 , 192.87, 194.56 (C1, C3, C11). Aromatic carbons C-6a, C-8, C-10a hidden
under solvent track signals.
9-Nitro-4-dedimethylamino doxycycline (4)
1g (2.49 mmol) of derivative 2 was dissolved in 15 mL of 97% H2SO4 and cooled at 0°C (ice bath). To
the stirring solution were added 1.1 equivalents of KNO3 (2.99 mmol, 0.3 grams) and the reaction
monitored via LC-MS. After 2 h the reaction was considered complete. The solution was diluted with
30 mL of MeOH and precipitated in 500 mL of ether at 0° C; the solid was then filtered and purified
through RP-MPLC.
Characterization Yield : 877 mg (79%) as a dark-yellow glas Analytical Data : C20H18N2O10 (MW = 446,37) APCI-MS m/z = 447.1 [M+1]+ HPLC : tr = 18.6 min. purity > 97 % (254nm)
Experimental Part
109
IR (film) : 3500-3100, 2933, 2878, 1615, 581, 1559, 1522, 1202 cm-1 1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.55 (d, 3H, J = 6.5 Hz, CH3 at C-6), 2.36 (ddd, 1H, J = 10.6, 5.4, 2.5 Hz, H-4a), 2.43 (dd,
1H, J = 13.2, 8.1 Hz, H-5a), 2.78 (dq, 1H, J = 13.5, 6.5 Hz, H-6), 2.92 (dd, 1H, J = 18.7, 2.2 Hz, H-4
alpha ), 3.04 (dd, 1H, J = 18.6, 5.2 Hz, 4-H beta), 3.65 (dd, 1H, J = 10.8, 8.3 Hz, H-5), 7.08 (d, 1H, J =
8.6 Hz, H-7), 8.10 (d, 1H, J = 8.6 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.17 (CH3), 31.04 (C-4), 40.42 (C-6), 44.82 (C-4a), 47.15 (C-5a), 69.77 (C-5), 76.02 (C-
12a), 99.58 (C-2), 107.94 (C-11a), 116.58 (C-10a), 119.01 (C-8), 132.44 (C-9), 138.03 (C-6a), 155.62
(C-10), 156.81 (C-7), 175.01, 177.46 (C-12, CONH2), 194.19 , 196.34 (C1, C3, C11).
9-Amino-4-dedimethylamino doxycycline (5)
1g (2.24 mmol) of derivative 4 was dissolved in 25 mL MeOH containing 0.1% of concentrated HCl
and 100 mg of 10% palladium on carbon. The mixture was hydrogenated in a Parr apparatus at 28° C
overnight, filtered through Celite to remove the catalyst, the solvent was then removed in vacuo and
the raw product purified through RP-MPLC.
Characterization Yield : 735 mg (78.9%) as grey solid Analytical Data : C20H20N2O8 (MW = 416,39) APCI-MS m/z = 417.0 [M+1]+ HPLC : tr = 11.8 min. purity > 97 % (254nm) IR (film) : 3470-3240, 2981, 2965, 2881, 1638, 1611, 1560, 1170 cm-1 1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.35 (ddd, 1H, J = 10.6, 5.3, 1.9 Hz, H-4a), 2.42 (dd,
1H, J = 12.5, 8.3 Hz, H-5a), 2.75 (dq, 1H, J = 12.8, 6.3 Hz, H-6), , 2.92 (dd, 1H, J = 18.3, 1.7 Hz, H-4
alpha ), 3.05 (dd, 1H, J = 18.3, 5.3 Hz, 4-H beta), 3.66 (dd, 1H, J = 10.6, 8.3 Hz, H-5), 7.04 (d, 1H, J =
8.3 Hz, H-8), 7.48 (d, 1H, J = 8.3 Hz, H-7).
13C NMR (90 MHz, CD3OD) :
Experimental Part
110
δ (ppm) = 16.21 (CH3), 30.99 (C-4), 39.89 (C-6), 44.95 (C-4a), 47.99 (C-5a), 69.88 (C-5), 75.97
(C-12a), 99.57 (C-2) 101.39, 108.03, 117.00, 117.67, 128.39 (5 Aromatic C), 146.93 (C-8), 154.55 (C-
6a), 159.10 (C-10), 175.08, 175.53 (C-12, CONH2), 194.94 , 196.45 (C1, C3, C11).
9-Iodo-4-dedimethylamino doxycycline (6)
1g (2.49 mmol) of derivative 2 was dissolved in 20 mL of TFA and put in an ice bath. To this stirring
solution were added 1.2 equivalents of N-Iodosuccinimide (2.99 mmol, 0.67 grams).The reaction
proceeded at 0° C for 30 min, then removed from the ice bath and allowed to react at r.t. for additional
5 h. TFA was removed in vacuo and 5 mL of MeOH were added to dissolve the residue.This solution
was precipitated in 500 mL of diethylether at 0° C, the solid filtered and purified through RP-MPLC.
Characterization Yield : 853 mg (65%) as a light-yellow solid Analytical Data : C20H18INO8 (MW = 527,27) APCI-MS m/z = 527.8 [M+1]+ HPLC : tr = 20.8 min. purity > 99 % (254nm) IR (film) : 3460-3200, 1644, 1602, 1566, 1414, 1136 cm-1 1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.50 (d, 3H, J = 6.5 Hz, CH3 at C-6), 2.35 (ddd, 1H, J = 10.7, 5.4, 2.6 Hz, H-4a), 2.39 (dd,
1H, J = 12.4, 7.8 Hz, H-5a), 2.67 (dq, 1H, J = 12.4, 6.4 Hz, H-6), 2.92 (dd, 1H, J = 18.7, 2.5 Hz, H-4
alpha ), 3.04 (dd, 1H, J = 18.6, 5.5 Hz, 4-H beta), 3.64 (dd, 1H, J = 10.9, 7.9 Hz, H-5), 6.75 (d, 1H, J =
8.2 Hz, H-7), 7.91 (d, 1H, J = 8.2 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.20 (CH3), 31.17 (C-4), 39.91 (C-6), 44.89 (C-4a), 47.74 (C-5a), 69.88 (C-5), 75.90
(C-12a), 83.81 (C-9), 99.59 (C-2), 107.86 (C-11a), 117.26 (C-10a), 118.79 (C-7), 146.67 (C-8), 149.96
(C-6a), 161.89 (C-10), 175.06, 175.81 (C-12, CONH2), 194.78 , 196.35 (C1, C3, C11).
Experimental Part
111
7,9-Diiodo-4-dedimethylamino doxycycline (7)
1g (1.89 mmol) of derivative 6 was dissolved in 20 mL of TFA and put in an ice bath. To this stirring
solution were added 1.2 equivalents of N-Iodosuccinimide (2.27 mmol, 0.51 grams).The reaction
proceeded at 0° C for 30 min, then removed from the ice bath and allowed to react at r.t. for additional
5 h. TFA was removed in vacuo and 5 mL of MeOH were added to dissolve the residue.This solution
was precipitated in 500 mL of diethylether at 0° C, the solid filtered and purified through RP-MPLC.
Characterization Yield : 1.02 g (82%) as a dark red-brown solid Analytical Data : C20H17I2NO8 (MW = 653,17) APCI-MS m/z = 653.9 [M+1]+ HPLC : tr = 21.7 min. purity 98 % (254nm) IR (film) : 3500-3200, 2981, 2967, 2870, 1730, 1644, 1566, 1407 cm-1 1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.23 (d, 3H, J = 7.4 Hz, CH3 at C-6), 2.23 (ddd, 1H, J = 11.0, 5.0, 2.0 Hz, H-4a), 2.48 (dd,
1H, J = 10.9, 2.4 Hz, H-5a), 2.81 (dd, 1H, J = 18.2, 1.9 Hz, H-4 alpha), 2.95 (dd, 1H, J = 18.1, 4.9 Hz,
4-H beta), 3.40 (dd, 1H, J = 10.9, 10.9 Hz, H-5), 3.86 (dq, 1H, J = 10.9, 7.7 Hz, H-6), 8.39 (s, 1H, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 19.54 (CH3), 30.21 (C-4), 39.66 (C-6), 43.96 (C-4a), 47.77 (C-5a), 68.04 (C-5), 75.85
(C-12a), 85.01 (C-9), 88.84 (C-7), 99.81 (C-2), 117.67 (C-10a), 156.16 (C-6a), 162.35 (C-8), 162.29,
162.35 (C-10, C-12), 174.67 (CONH2), 195.13 , 197.11, 200.02 (C1, C3, C11).
11a-Bromo-4-dedimethylamino doxycycline (10)
Experimental Part
112
500mg (1.24 mmol) of derivative 2 were dissolved in 10 mL of CHCl3, 1.2 equivalents of N-
Bromosuccinimide (MW = 177.98, 1.49 mmol, 264.8 mg) were added and the reaction stirred
overnight at room temperature. The solvent was removed in vacuo, the remaining solid was dissolved
in methanol and purified through RP-MPLC.
Characterization Yield : 130.9 mg (22%)as pale yellow powder Analytical Data : C20H18BrNO8 (MW = 480,27) APCI-MS m/z = 481.7 [M+1]+ HPLC : tr = 17.9 min. purity 92 % (254nm) IR (film) : 3550-3100, 2973, 2929, 2875, 1731, 1646, 1601, 1567, 1446, 195, 1051 cm-1 1H NMR (360 MHz, Methanol d-4) :
δ (ppm) = 1.51 (d, 3H, J = 6.7 Hz, CH3 at C-6), 2.35 (ddd, 1H, J = 11.0, 5.3, 2.6 Hz, H-4a), 2.70 (dq,
1H, J = 13.7, 6.5 Hz, H-6), 2.86 (dd, 1H, J = 10.1, 1.6 Hz, H-5a), 2.91 (dd, 1H, J = 18.6, 2.6 Hz, H-4
alpha ), 3.04 (dd, 1H, J = 18.6, 5.5 Hz, 4-H beta), 3.64 (dd, 1H, J = 10.6, 8.0 Hz, H-5), 6.81 (d, 1H, J =
8.4 Hz, H-9), 6.92 (d, 1H, J = 7.7 Hz, H-7), 7.45 (t, 1H, J = 7.9 Hz, H-8).
13C NMR (150 MHz, Methanol d-4) :
δ (ppm) = 16.29 (CH3), 25.07 (C-4), 35.48 (C-6), 40.11 (C-4a), 44.97 (C-5a), 59.56 (C-11a), 69.93 (C-
5), 75.83 (C-12a), 99.59 (C-2), 108.01, 117.02, 121.48, 137.55, 139.41, 149.48 (Aromatic Cs), 163.45
(C-10), 175.00, 175.07 (C-12, CONH2), 195.49 , 195.54, 196.38 (C1, C3, C11).
9,11a-Dibromo-4-dedimethylamino doxycycline (9)
1 gram (2.49 mmol) of derivative 2 was dissolved in 10 mL of trfluoroacetic acid, 2.2 equivalents of N-
Bromosuccinimide (MW = 177.98, 5.48 mmol, 975.1 mg) were added and the reaction stirred at room
temperature for 3 hours. TFA was removed in vacuo and the remaining oil diluted with methanol and
purified through RP-MPLC.
Characterization Yield : 975 mg (69.6%) as dark brown oil Analytical Data : C20H19Br2NO8 (MW = 559,16)
Experimental Part
113
APCI-MS m/z = 561.9 [M+2]+ HPLC : tr = 20.0 min. purity 97 % (254nm) IR (film) : 3500-3130, 2981, 2873, 1739, 1647, 1600, 1566, 1445, 1191, 1066, 1032 cm-1 1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.63 (d, 3H, J = 7.5 Hz, CH3 at C-6), 2.35 (ddd, 1H, J = 11.3, 4.9, 1.5 Hz, H-4a), 2.81 (br dd,
1H, J = 18.1 Hz, H-4 alpha ), 2.93 (dd, 1H, J = 10.2, 1.5 Hz, H-5a), 2.98 (br dd, 1H, J = 18.1 Hz, 4-H
beta), 3.30 (dd, 1H, J = 10.6, 10.6 Hz, H-5), 4.32 (dq, 1H, J = 7.5, 1.5 Hz, H-6), 6.78 (d, 1H, J = 9.0
Hz, H-7), 7.74 (d, 1H, J = 9.0 Hz, H-8). H at C-12 hidden under methanol peak (δ = 3.34 ppm).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 21.59 (CH3), 32.22 (C-4), 35.53 (C-6), 46.41 (C-4a), 54.54 (11a), 59.67 (C-5a), 68.20 (12),
69.44 (C-5), 82.25 (C-12a), 99.36 (C-2), 114.10(C-9), 116.74(C-10a), 119.66 (C-7), 143.00 (C-8),
147.44 (C-6a), 163.47 (C-10), 174.57 (CONH2), 191.32 (C-11), 195.24 , 197.06 (C1, C3).
9-Bromo-4-dedimethylamino doxycycline (8)
To 500 mg (0.89 mmol) of derivative 9 in water/DMF (5+5 mL) were added 3 equivalents of sodium
dithionite (MW = 174.09, 2.68 mmol, 467 mg) and the reaction monitored via LC-MS. After 3 h the
reaction was considered complete. The solution was freeze-dried and the crude product purified
through RP-HPLC.
Characterization Yield : 125 mg (29%) as pale yellow powder Analytical Data : C20H19Br2NO8 (MW = 480,27) APCI-MS m/z = 481.9 [M+1]+ HPLC : tr = 18.4 min. purity 85 % (254nm) IR (film) : 3570-3100, 2973, 2874, 1637, 1560, 1539, 1447, 1291, 1196, 1051 cm-1 1H NMR (360 MHz, Acetone d-6) :
δ (ppm) = 1.32 (d, 3H, J = 7.3 Hz, CH3 at C-6), 2.34 (ddd, 1H, J = 10.9, 4.8, 1.5 Hz, H-4a), 2.66 (dd,
1H, J = 10.9, 1.6 Hz, H-5a), 2.89 (dd, 1H, J = 18.2, 1.6 Hz, H-4 alpha ), 2.98 (dd, 1H, J = 18.1, 5.2 Hz,
Experimental Part
114
4-H beta), 3.59 (dd, 1H, J = 10.9, 10.9 Hz, H-5), 4.07 (dq, 1H, J = 7.2, 1.6 Hz, H-6), 6.77 (d, 1H, J =
8.8 Hz, H-7), 7.71 (d, 1H, J = 8.9 Hz, H-8).
9-tert-butyl-4-dedimethylamino doxycycline (11)
A solution of 4-DDMA-Doxycycline (100 mg, 0.25 mmol) in 2 mL of tert-butanol and 3 mL of
methanesulfonic acid was stirred at room temperature for 18 hours. After evaporation of the solvents
in vacuo, the crude product was dissolved in methanol and purified through reverse phase preparative
HPLC to afford the pure compound.
Characterization Yield : 80.9 mg (71%) Analytical Data : C24H27NO8 (MW = 457,48) APCI-MS m/z = 459.2 [M+2]+ HPLC : tr = 18.8 min. purity 95 % (254nm) IR (film) : 3500-3160, 2955, 2903, 2873, 1667, 1598, 1561, 1420 cm-1 1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.40 (s, 9H, 3 CH3),1.49 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.32 (together with H-4a, 1H, J =
7.7, H-5a), 2.33 (ddd, 1H, J = 10.7, 5.6, 2.5 Hz, H-4a), 2.63 (dq, 1H, J = 13.1, 6.6 Hz, H-6), 2.92 (dd,
1H, J = 18.6, 2.5 Hz, H-4 alpha ), 3.04 (dd, 1H, J = 18.6, 5.4 Hz, 4-H beta), 3.62 (dd, 1H, J = 10.7, 7.9
Hz, H-5), 6.83 (d, 1H, J = 7.9 Hz, H-7), 7.46 (t, 1H, J = 7.9 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.68 (CH3), 30.28 (tBu CH3), 31.82 (C-4), 36.11 (tBu C-CH3), 40.34 (C-6), 45.42 (C-4a),
48.24 (C-5a), 70.38 (C-5), 76.30 (C-12a), 100.10 (C-2), 108.77 (C-11a), 116.35, 118.98, 135.13,
137.78 (4 Aromatic C), 147.53 (C-6a), 163.52 (C-10), 174.82, 175.60 (C-12, CONH2), 195.16, 196.89,
196.92 (C1, C3, C11).
Experimental Part
115
General procedure for acylation derivatives : 0.100 grams of derivative 5 (9-amino-4-dedimethylamino doxycycline) were dissolved in 3 mL of dry
DMF. To this solution were added 2 equivalents of NaHCO3 and 2 equivalents of the appropriate
acylating agent. The reaction proceeded at room temperature for 1-3 hours. After the reaction was
considered complete, the solvent was removed in vacuo, the product solubilized in methanol and
purified via RP-HPLC, yielding the desired pure product.
9-Acetylamino-4-dedimethylamino doxycycline (12)
78.4 mg (0.188 mmol) of derivative 5 were dissolved in 3 mL dry DMF and 2 equivalents of NaHCO3
(MW = 84, 0.376 mmol, 31.5 mg) were added. Then, 2 equivalents of acetic anhydride (MW = 102,
0.376 mmol, 38.4 mg) were injected. After 1 hour the reaction was considered teminated, according to
LC-MS analysis. The solvent was removed in vacuo and the raw product was purified through reverse
phase HPLC.
Characterization Yield : 56 mg (65%) as a light yellow glas Analytical Data : C22H22N2O9 (MW = 458,43) APCI-MS m/z = 459.5 [M+1]+ HPLC : tr = 15.1 min. purity > 97 % (254nm) IR (film) : 3560-3100, 2976, 2874, 1754, 1672, 1610, 1526, 1241 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.51 (d, 3H, J = 6.7 Hz, CH3 at C-6), 2.19 (s, 3H, CH3 acetyl), 2.34 (ddd, 1H, J = 10.9, 5.0,
1.9 Hz, H-4a), 2.37 (dd, 1H, J = 12.5, 7.9 Hz, H-5a), 2.66 (dq, 1H, J = 13.0, 6.5 Hz, H-6), 2.92 (dd, 1H,
J = 18.3, 1.9 Hz, H-4 alpha ), 3.04 (dd, 1H, J = 18.5, 5.0 Hz, 4-H beta), 3.63 (dd, 1H, J = 10.6, 7.9 Hz,
H-5), 6.90 (d, 1H, J = 8.3 Hz, H-7), 8.14 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.20 (CH3), 23.70 (CH3 acetyl), 31.32 (C-4), 39.79 (C-6), 44.96 (C-4a), 49.92 (C-5a), 69.96
(C-5), 75.90 (C-12a), 99.60 (C-2), 108.04, 116.12, 116.78, 126.57, 129.68 (5 Aromatic C), 144.66 (C-
6a), 153.84 (C-10), 172.00 (C=O acetyl), 175.07, 175.43 (C-12, CONH2), 195.50, 196.39 (C1, C3,
C11).
Experimental Part
116
9-Propionylamino-4-dedimethylamino doxycycline (13)
To 100 mg (0.24 mmol) of derivative 5 dissolved in 3 mL of dry DMF 2 equivalents of NaHCO3 (MW =
84, 0.48 mmol, 40.4 mg) and 2 equivalents of propionic anhydride (MW = 130.14, 0.48 mmol, 62.6
mg) were added. After 2 hour the reaction was considered teminated, according to LC-MS analysis.
The solvent was removed in vacuo and the raw product was purified through reverse phase HPLC.
Characterization Yield : 72 mg (64%) as light yellow glas Analytical Data : C23H24N2O9 (MW = 472,46) APCI-MS m/z = 473.6 [M+1]+ HPLC : tr = 16.4 min. purity > 97 % (254nm) IR (film) : 3500-3100, 2977, 2877, 1748, 1660, 1609, 1563, 1523, 1426, 1241 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.23 (t, 3H, J = 7.6 Hz, CH3 propionyl), 1.52 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.36 (ddd, 1H, J
= 11.0, 5.4, 2.1 Hz, H-4a), 2.38 (dd, 1H, J = 12.5, 7.9 Hz, H-5a), 2.49 (q, 2H, J = 7.6, CH2 propionyl),
2.68 (dq, 1H, J = 13.2, 6.6 Hz, H-6), 2.94 (dd, 1H, J = 18.3, 1.9 Hz, H-4 alpha ), 3.05 (dd, 1H, J = 18.3,
5.0 Hz, 4-H beta), 3.65 (dd, 1H, J = 10.6, 7.9 Hz, H-5), 6.91 (d, 1H, J = 8.3 Hz, H-7), 8.17 (d, 1H, J =
8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 10.21 (CH3 propionyl), 16.18 (CH3), 30.84 (CH2 propionyl, C-4), 39.79 (C-6), 44.93 (C-4a),
48.13 (C-5a), 69.93 (C-5), 75.89 (C-12a), 101.39 (C-2), 108.04, 116.12, 116.76, 126.61, 129.55 (5
Aromatic C), 144.52 (C-6a), 153.80 (C-10), 175.06, 175.43, 175.61 (C=O propionyl, C-12, CONH2),
195.51, 196.39 (C1, C3, C11).
9-Benzoylamino-4-dedimethylamino doxycycline (14)
Experimental Part
117
100 mg (0.24 mmol) of derivative 5 were dissolved in 3 mL dry DMF and 2 equivalents of NaHCO3
(MW = 84, 0.48 mmol, 40.4 mg) plus 2 equivalents of benzoic anhydride (MW = 226.23, 0.48 mmol,
109 mg) were added. After 2 hour the reaction was considered teminated, according to LC-MS
analysis. The solvent was removed in vacuo and the raw product was purified through reverse phase
HPLC.
Characterization Yield : 31 mg (25%) as yellow solid Analytical Data : C27H24N2O9 (MW = 520,50) APCI-MS m/z = 421.8 [M+1]+ HPLC : tr = 19.2 min. purity > 97 % (254nm) IR (film) : 3530-3160, 3070, 2975, 2873, 1744, 1679, 1660, 1651, 1523, 1240, 1055 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.36 (ddd, 1H, J = 10.7, 5.4, 2.1 Hz, H-4a), 2.41 (dd,
1H, J = 12.5, 7.9 Hz, H-5a), 2.71 (dq, 1H, J = 13.2, 6.6 Hz, H-6), 2.93 (dd, 1H, J = 18.3, 2.1 Hz, H-4
alpha ), 3.05 (dd, 1H, J = 18.5, 5.3 Hz, 4-H beta), 3.65 (dd, 1H, J = 10.6, 7.9 Hz, H-5), 6.98 (d, 1H, J =
8.3 Hz, H-7), 7.53 (t, 2H, J = 7.7 Hz, H-3’/5’), 7.60 (t, 1H, J = 7.4 Hz, H-4’), 7.95 (t, 2H, J = 7.4 Hz, H-
2’/6’), 8.22 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.24 (CH3), 31.17 (C-4), 39.88 (C-6), 45.00 (C-4a), 48.16 (C-5a), 69.99 (C-5), 75.92 (C-
12a), 99.59 (C-2), 108.06 (C-11a), 116.33, 116.99, 126.46, 128.49, 129.57, 129.82, 130.13, 133.16,
135.78 (10 Aromatic C), 145.29 (C-6a), 154.44 (C-10), 168.35 (C=O benzoyl), 175.09, 175.62 (C-12,
CONH2), 195.42, 196.38 (C1, C3, C11).
9-Pivaloylamino-4-dedimethylamino doxycycline (15)
To 100 mg (0.24 mmol) of derivative 5 dissolved in 3 mL of dry NMP 2 equivalents of NaHCO3 (MW =
84, 0.48 mmol, 40.4 mg) and 2 equivalents of trimethylacetyl chloride (MW = 120.58, 0.48 mmol, 57.9
mg, 0.059 mL) were added. After 2 hour the reaction was considered teminated, according to LC-MS
analysis. The solvent was removed in vacuo and the raw product was purified through reverse phase
HPLC.
Experimental Part
118
Characterization Yield : 50 mg (42%) Analytical Data : C25H28N2O9 (MW = 500,51) APCI-MS m/z = 501.1 [M+1]+ HPLC : tr = 13.3 min. purity > 97 % (254nm) IR (film) : 3540-3130, 2972, 2947, 2880, 1676, 1610, 1562, 1431, 1200, 1179, 1134 cm-1 1H NMR (360 MHz, CD3OD) :
δ (ppm) = 0.94 (s, 9H, ((CH3)3), 1.57 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.37 (ddd, 1H, J = 10.6, 5.4, 2.4
Hz, H-4a), 2.47 (dd, 1H, J = 12.5, 8.1 Hz, H-5a), 2.82 (dq, 1H, J = 13.2, 6.4 Hz, H-6), , 2.92 (dd, 1H, J
= 18.6, 2.2 Hz, H-4 alpha ), 3.05 (dd, 1H, J = 18.7, 5.5 Hz, 4-H beta), 3.67 (dd, 1H, J = 10.6, 8.1 Hz,
H-5), 7.22 (d, 1H, J = 8.6 Hz, H-7), 7.80 (d, 1H, J = 8.6 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 17.21 (CH3), 20.61 ((CH3)3), 30.91 (C-4), 41.19 (C-6, C-(CH3)3), 45.85 (C-4a), 48.36 (C-5a),
62.18 (C-5), 70.77 (C-12a), 99.69, 108.93, 119.20, 119.75, 125.54 (5 Aromatic C), 152.83 (C-6a),
156.76 (C-10), 176.12, 178.73 (NH-C=O, C-12, CONH2), 194.35 , 195.25, 197.3(C1, C3, C11).
Experimental Part
119
General conditions for the N-alkyl derivatives : To a solution of the amine 5 in aqueous MeOH (50 %) aldehyde or ketone (1-10 equiv), NaCNBH3 (1-
2 equiv) and HCl (1 equiv) were added. The reaction mixture was stirred at room temperature for 1.5
h, then the product was purified by reversed-phase MPLC or HPLC, obtaining pure substances as
TFA salts.
9-Dimethylamino-4-dedimethylamino doxycycline (16)
To a solution of the amine 5 (0.100g, 0.24 mmol) in 10 mL of 50% aqueous MeOH, formaldehyde (4
equivalents, 0.96 mmol, MW = 30, 28.8 mg, 0.018 mL), NaCNBH3 (1.5 equivalents, 0.36 mmol, MW =
62.84, 22.6 mg) and 0.03 mL of concentrated HCl were added. The reaction mixture was stirred at
room temperature for 1.5 h, then the product was isolated by reversed-phase HPLC.
Characterization Yield : 59 mg (56%) as yellow glas Analytical Data : C22H24N2O8 (MW = 444,45) APCI-MS m/z = 446.1 [M+1]+ HPLC : tr = 10.5 min. purity > 99 % (254nm) IR (film) : 3540-3160, 2979, 2875, 1680, 1614, 1558, 1432, 1201 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.55 (d, 3H, J = 6.7 Hz, CH3 at C-6), 2.36 (ddd, 1H, J = 10.6, 5.6, 2.2 Hz, H-4a), 2.44 (dd,
1H, J = 12.5, 8.3 Hz, H-5a), 2.79 (dq, 1H, J = 13.4, 6.6 Hz, H-6), 2.91 (dd, 1H, J = 18.1, 2.0 Hz, H-4
alpha ), 3.04 (dd, 1H, J = 18.1, 5.5 Hz, 4-H beta), 3.30 (s, 6H, N-(CH3)2), 3.67 (dd, 1H, J = 10.6, 8.3
Hz, H-5), 7.17 (d, 1H, J = 8.7 Hz, H-7), 7.86 (d, 1H, J = 8.7 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.18 (CH3), 31.39 (C-4), 40.03 (C-6), 44.92 (C-4a), 46.00 (N-CH3), 47.53 (C-5a), 69.86 (C-
5), 76.06 (C-12a), 99.55 (C-2), 107.98 (C-11a), 117.93, 118.53, 128.11, 129.60 (4 Aromatic C),
151.61, 154.35 (C-9, C-10), 175.05, 177.62 (C-12, CONH2), 194.33, 196.44 (C1, C3, C11).
Experimental Part
120
9-Diethylamino-4-dedimethylamino doxycycline (17)
To a solution of the amine 5 (0.160g, 0.384 mmol) in 10 mL of 50% aqueous MeOH, acetaldehyde (2
equivalents, 0.77 mmol, MW = 44.05, 33.8 mg, 0.043 mL), NaCNBH3 (1.5 equivalents, 0.576 mmol,
MW = 62.84, 36.3 mg) and 0.04 mL of concentrated HCl were added. The reaction mixture was stirred
at room temperature for 2 h, then the product was isolated by reversed-phase HPLC.
Characterization Yield : 72 mg (39%) as light yellow glas Analytical Data : C24H28N2O8 (MW = 472,50) APCI-MS m/z = 473.1 [M+1]+ HPLC : tr = 11.8 min. purity > 99 % (254nm) IR (film) : 3460-3200, 2987, 2879, 1672, 1559, 1434, 1201, 1176, 1134 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.15 (t, 6H, J = 7.1 Hz, CH3 ethyl), 1.58 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.38 (ddd, 1H, J =
10.7, 5.4, 2.3 Hz, H-4a), 2.49 (dd, 1H, J = 12.5, 8.0 Hz, H-5a), 2.82 (dq, 1H, J = 13.0, 6.7 Hz, H-6),
2.92 (dd, 1H, J = 18.1, 2.4 Hz, H-4 alpha ), 3.05 (dd, 1H, J = 18.1, 5.4 Hz, 4-H beta), 3.69 (m, 5H, H-5
+ 2 CH2 ethyl), 7.24 (d, 1H, J = 8.6 Hz, H-7), 7.81 (d, 1H, J = 8.6 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 10.43 (CH3 ethyl), 16.17 (CH3), 31.39 (C-4), 40.09 (C-6), 44.93 (C-4a), 47.47 (C-5a), 50.17
(CH2 ethyl), 69.88 (C-5), 76.08 (C-12a), 99.52 (C-2), 107.99 (C-11a), 118.38, 118.55, 123.61, 129.63
(4 Aromatic C), 152.04, 155.93 (C-9, C-10), 175.07, 177.85 (C-12, CONH2), 194.26, 196.42 (C1, C3,
C11).
9-Dipropylamino-4-dedimethylamino doxycycline (18)
Experimental Part
121
To a solution of the amine 5 (0.100g, 0.24 mmol) in 10 mL of 50% aqueous MeOH, propyl aldehyde (4
equivalents, 0.96 mmol, MW = 58.08, 55.8 mg, 0.070 mL), NaCNBH3 (1.5 equivalents, 0.36 mmol,
MW = 62.84, 22.6 mg) and 0.03 mL of concentrated HCl were added. The reaction mixture was stirred
at room temperature for 1 hour, and then the product was isolated by reversed-phase HPLC.
Characterization Yield : 53 mg (44%) as yellow glas Analytical Data : C26H32N2O8 (MW = 500,55) APCI-MS m/z = 501.4 [M+1]+ HPLC : tr = 13.3 min. purity > 99 % (254nm) IR (film) : 3500-3200, 2965, 2876, 1698, 1672, 1559, 456, 1434, 1201, 1176, 1057 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 0.93 (t, 6H, J = 7.4 Hz, CH3 propyl), 1.50 (m, 4H, CH3-CH2 propyl), 1.57 (d, 3H, J = 6.8 Hz,
CH3 at C-6), 2.37 (ddd, 1H, J = 10.7, 5.5, 2.3 Hz, H-4a), 2.47 (dd, 1H, J = 12.5, 8.0 Hz, H-5a), 2.82
(dq, 1H, J = 13.0, 6.7 Hz, H-6), 2.93 (dd, 1H, J = 18.3, 2.3 Hz, H-4 alpha ), 3.05 (dd, 1H, J = 18.3, 5.2
Hz, 4-H beta), 3.58 (br s, 4H, N-CH2), 3.67 (dd, 1H, J = 10.8, 8.1 Hz, H-5), 7.22 (d, 1H, J = 8.7 Hz, H-
7), 7.81 (d, 1H, J = 8.7 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 10.43 (CH3 ethyl), 16.17 (CH3), 19.47 (N-CH2-CH2), 31.39 (C-4), 40.09 (C-6), 44.93 (C-4a),
47.46 (C-5a), 61.14 (N-(CH2)2), 69.88 (C-5), 76.06 (C-12a), 99.56 (C-2), 107.93 (C-11a), 118.35,
118.48, 124.74, 129.39 (4 Aromatic C), 151.81, 155.65 (C-9, C-10), 175.07, 177.87 (C-12, CONH2),
194.28, 196.44, 196.91 (C1, C3, C11).
9-Isopropylamino-4-dedimethylamino doxycycline (19)
To a solution of the amine 5 (0.100g, 0.24 mmol) in 10 mL of 50% aqueous MeOH, acetone (2
equivalents, 0.48 mmol, MW = 58.08, 27.9 mg, 0.035 mL), NaCNBH3 (2 equivalents, 0.48 mmol, MW
= 62.84, 30 mg) and 0.03 mL of concentrated HCl were added. The reaction mixture was stirred at
room temperature for 1 hour, and then the product was isolated by reversed-phase HPLC.
Characterization
Experimental Part
122
Yield : 83 mg (75%) as yellow powder Analytical Data : C23H26N2O8 (MW = 458,47) APCI-MS m/z = 460.2 [M+1]+ HPLC : tr = 14.5 min. purity > 99 % (254nm) IR (film) : 3500-3200, 2980, 2874, 1734, 1692, 1681, 1556, 1245, 1201, 1132, 1052 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.34 (m, 6H, CH3 isopropyl), 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.36 (ddd, 1H, J = 10.7,
5.5, 2.3 Hz, H-4a), 2.43 (dd, 1H, J = 12.5, 8.0 Hz, H-5a), 2.76 (dq, 1H, J = 13.1, 6.7 Hz, H-6), 2.92 (dd,
1H, J = 18.3, 2.2 Hz, H-4 alpha ), 3.05 (dd, 1H, J = 18.5, 5.2 Hz, 4-H beta), 3.66 (dd, 1H, J = 10.5, 8.1
Hz, H-5), 3.83 (q, 1H, J = 6.4 Hz, CH isopropyl), 7.08 (d, 1H, J = 8.3 Hz, H-7), 7.43 (d, 1H, J = 8.3 Hz,
H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.19 (CH3), 20.14 (NH-CH-CH3), 30.94 (C-4), 39.95 (C-6), 44.94 (C-4a), 47.84 (C-5a),
50.61 (CH isopropyl), 69.87 (C-5), 76.02 (C-12a), 99.64 (C-2), 108.08 (C-11a), 117.07, 117.97,
128.57, 137.51 (4 Aromatic C), 154.77, 162.48 (C-9, C-10), 175.07, 176.86 (C-12, CONH2), 194.43,
194.82, 196.43 (C1, C3, C11).
9-Cyclopentylamino-4-dedimethylamino doxycycline (20)
To a solution of the amine 5 (0.100g, 0.24 mmol) in 10 mL of 50% aqueous MeOH, cyclopentanone
(1.2 equivalents, 0.29 mmol, MW = 84.12, 24.3 mg, 0.0255 mL), NaCNBH3 (1.5 equivalents, 0.36
mmol, MW = 62.84, 22.7 mg) and 0.03 mL of concentrated HCl were added. The reaction mixture was
stirred at room temperature for 2 hours, and then the product was isolated by reversed-phase HPLC.
Characterization Yield : 68 mg (58%%) as brown-yellow solid Analytical Data : C25H28N2O8 (MW = 484,51) APCI-MS m/z = 486.2 [M+1]+ HPLC : tr = 19.6 min. purity > 99 % (254nm)
Experimental Part
123
IR (film) : 3520-3250, 2965, 2874, 1673, 1610, 1564, 1494, 1447, 1241, 1199, 1134, 1052 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.53 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.6–1.75 (m, 4H, 2 CH2-CH2-CH cyclopentyl), 1.81-
1.88 (m, 2H) and 1.99-2.08 (m, 2H) (CH2-CH2-CH and CH2-CH2-CH cyclopentyl), 2.35 (ddd, 1H, J =
10.7, 5.4, 2.1 Hz, H-4a), 2.41 (dd, 1H, J = 12.5, 8.1 Hz, H-5a), 2.72 (dq, 1H, J = 13.1, 6.4 Hz, H-6),
2.92 (dd, 1H, J = 18.3, 2.1 Hz, H-4 alpha ), 3.04 (dd, 1H, J = 18.3, 5.0 Hz, 4-H beta), 3.65 (dd, 1H, J =
10.7, 8.1 Hz, H-5), 4.00 (m, 1H, CH cyclopentyl), 7.02 (d, 1H, J = 8.3 Hz, H-7), 7.36 (d, 1H, J = 8.3 Hz,
H-8).
13C NMR (150 MHz, CD3OD) :
δ (ppm) = 16.20 (CH3), 24.86 (CH2-CH2-CH cyclopentyl), 31.79, 31.83 (C-4, CH2-CH2-CH cyclopentyl),
39.87 (C-6), 44.94 (C-4a), 47.99 (C-5a), 61.33 (CH cyclopentyl), 69.88 (C-5), 75.97 (C-12a), 99.51 (C-
2), 108.08 (C-11a), 117.17, 117.58, 126.59, 127.65 (4 Aromatic C), 145.97, 154.09 (C-9, C-10),
175.08, 176.49 (C-12, CONH2), 195.05, 196.42 (C1, C3, C11).
9-Cyclohexylamino-4-dedimethylamino doxycycline (21)
To a solution of the amine 5 (0.100g, 0.24 mmol) in 10 mL of 50% aqueous MeOH, cyclohexanone
(1.1 equivalents, 0.264 mmol, MW = 98.14, 25.9 mg, 0.0274 mL), NaCNBH3 (1.5 equivalents, 0.36
mmol, MW = 62.84, 22.7 mg) and 0.03 mL of concentrated HCl were added. The reaction mixture was
stirred at room temperature for 2 hours, and then the product was isolated by reversed-phase HPLC.
Characterization Yield : 56 mg (47%) as dark-yellow glas Analytical Data : C26H30N2O8 (MW = 498,54) APCI-MS m/z = 499.2 [M+1]+ HPLC : tr = 18.4 min. purity > 99 % (254nm) IR (film) : 3530-3270, 2980, 2935, 2859, 1674, 1610, 1561, 1454, 1238, 1201, 1178, 1135 cm-1 1H NMR (600 MHz, CD3OD) :
Experimental Part
124
δ (ppm) = 1.31-1.47 (m, 5H, 2 CH2 pos. 3’ and 5’ + 1H pos. 4’ cyclohexyl), 1.54 (d, 3H, J = 6.8 Hz, CH3
at C-6), 1.66-1.74 (m, 1H, 1H position 4’ cyclohexyl), 1.82-1.88 and 2.01-2.08 (2 m, each 2H, CH2 pos.
2’ and 6’ cyclohexyl), 2.35 (ddd, 1H, J = 10.5, 5.2, 2.1 Hz, H-4a), 2.42 (dd, 1H, J = 12.5, 8.1 Hz, H-5a),
2.74 (dq, 1H, J = 13.0, 6.4 Hz, H-6), 2.92 (dd, 1H, J = 18.3, 2.0 Hz, H-4 alpha ), 3.05 (dd, 1H, J = 18.5,
5.1 Hz, 4-H beta), 3.43-3.49 (m, 1H, CH 1’ cyclohexyl), 3.66 (dd, 1H, J = 10.5, 8.2 Hz, H-5), 7.04 (d,
1H, J = 8.7 Hz, H-7), 7.38 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.15 (CH3), 25.67, 26.33 (C-3’, C-5’, C-6’), 31.68 (C-4, C-2’, C-4’), 39.89 (C-6), 44.96 (C-
4a), 47.96 (C-5a), 59.27 (C-1’), 69.91 (C-5), 75.98 (C-12a), 99.58 (C-2), 107.98 (C-11a), 117.11,
117.74, 127.28, 140.66 (4 Aromatic C), 146.55, 154.34 (C-9, C-10), 175.08, 176.57 (C-12, CONH2),
194.95, 194.21, 196.42 (C1, C3, C11).
9-Isopropyl(methyl)-amino-4-dedimethylamino doxycycline (22)
To a solution of the secondary amine 19 (0.30 g, 0.065 mmol) in 5 mL of 50% aqueous MeOH,
formaldehyde (37% in water, 0.5 mL), NaCNBH3 (1.5 equivalents, 0.09 mmol, MW = 62.84, 6 mg) and
0.01 mL of concentrated HCl were added. The reaction mixture was stirred at room temperature for
1.5 h, and then the product was isolated by reversed-phase HPLC.
Characterization Yield : 15 mg (48%) Analytical Data : C24H28N2O8 (MW = 472,50) APCI-MS m/z = 474.1 [M+1]+ HPLC : tr = 11.0 min. purity > 99 % (254nm) IR (film) : 3500-3200, 2977, 2935, 2875, 1679, 1613, 1566, 1455, 1428, 1247, 1200, 1182,
1135, 1065 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.37 (m, 6H, CH3 isopropyl), 1.56 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.36 (ddd, 1H, J = 10.7,
5.5, 2.1 Hz, H-4a), 2.46 (dd, 1H, J = 12.5, 8.0 Hz, H-5a), 2.80 (dq, 1H, J = 13.1, 6.4 Hz, H-6), 2.92 (dd,
1H, J = 18.3, 2.0 Hz, H-4 alpha ), 3.05 (dd, 1H, J = 18.5, 5.2 Hz, 4-H beta), 3.27 (s, 3H, CH3-N), 3.67
Experimental Part
125
(dd, 1H, J = 10.5, 8.1 Hz, H-5), 4.07 (m, 1H, CH isopropyl), 7.18 (d, 1H, J = 8.7 Hz, H-7), 7.81 (d, 1H,
J = 8.7 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.15 (CH3), 17.99 (CH3 isopropyl), 31.16 (C-4), 40.03, 40.26 (C-6, N-CH3), 44.92 (C-4a),
47.51 (C-5a), 63.00 (CH isopropyl), 69.85 (C-5), 76.07 (C-12a), 99.53 (C-2), 107.98 (C-11a), 117.78,
118.53, 127.51, 129.81 (4 Aromatic C), 151.60, 154.94 (C-9, C-10), 175.05, 177.69 (C-12, CONH2),
193.41, 194.38, 196.41 (C1, C3, C11).
Experimental Part
126
General procedure for 4-ddma- doxycycline alkyne derivatives; 1 equivalent of 9-iodo-4-dedimethylamino doxycycline, 10% of tetrakistriphenylphosphine palladium(0)
catalyst and 10% of CuI were dissolved in dry THF. Triethylamine (5 equivalents) and 3-5 equivalents
of alkyne were added and the mixture was vigorously stirred between room temperature and 70 °C for
2-24 h. Filtration through Celite and removal of the solvent in vacuo produced crude products, which
were then purified through preparative reverse phase HPLC.
9-Phenylethynyl-4-dedimethylamino doxycycline (23)
263 mg (0.5 mmol) of derivative 6, 0.0577 g (0.05 mmol) of Tetrakis(triphenylphosphine)palladium(0),
0.0095 g (0.05 mmol) of copper iodide, 0.33 ml (2 mmol) of phenylacetylene were dissolved in 5 mL of
dry THF. The reaction mixture was charged with N2 and then 0.7 mL of TEA (5 mmol) were added via
syringe. After 2 hours the reaction was considered complete according to LC-MS analysis. The
catalyst was removed by filtration through Celite, THF removed in vacuo and the crude product thus
obtained dissolved in acetonitrile. Purification via reverse phase HPLC afforded the pure compound.
Characterization Yield : 1.074 g (42%) as yellow-brown glas Analytical Data : C28H23NO8 (MW = 501,50) APCI-MS m/z = 502.0 [M+1]+ HPLC : tr = 21.8 min. purity > 97 % (254nm) IR (film) : 3500-3200, 3065, 2978, 2875, 1650, 1604, 1577, 1555, 1424, 1279, 1241, 1201,
756 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.52 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.36 (ddd, 1H, J = 10.7, 5.3, 2.4 Hz, H-4a), 2.41 (dd,
1H, J = 12.5, 8.0 Hz, H-5a), 2.73 (dq, 1H, J = 13.0, 6.5 Hz, H-6), 2.92 (dd, 1H, J = 18.5, 2.4 Hz, H-4
alpha ), 3.05 (dd, 1H, J = 18.5, 5.5 Hz, 4-H beta), 3.65 (dd, 1H, J = 10.7, 8.0 Hz, H-5), 6.95 (d, 1H, J =
8.3 Hz, H-7), 7.32-7.40 (m, 3H, H at C2’,4’,6’), 7.48-7.55 (m, 2H, H at C3’,5’), 7.62 (d, 1H, J = 7.9 Hz,
H-8).
13C NMR (90 MHz, CD3OD) :
Experimental Part
127
δ (ppm) = 16.22 (CH3), 31.16 (C-4), 40.17 (C-6), 44.95 (C-4a), 47.80 (C-5a), 69.94 (C-5), 75.91 (C-
12a), 85.17 (C1’ alkyne), 95.01 (C2’ alkyne), 99.62 (C-2), 108.01 (C-11a), 112.45 (C-9), 116.81,
117.12 (C-10a, C-7), 124.74, 129.41, 129.50, 132.51 (6 Aromatic C), 140.32 (C-8), 149.98 (C-6a),
163.63 (C-10), 175.08, 175.70, (C-12, CONH2), 195.24, 196.37 (C1, C3, C11).
4-((6R,7S,11aS)-10-Carbamoyl-7,9,11a,12-tetrahydroxy-6-methyl-11,13-dioxo-6,6a,7,7a,8,11,11a,13-octahydrotetraceno[1,2-b]furan-2-yl)butanoic acid (24)
132 mg (0.25 mmol) of derivative 6, 0.0289 g (0.025 mmol) of
Tetrakis(triphenylphosphine)palladium(0), 0.0047 g (0.025 mmol) of copper iodide, 0.11 ml (1 mmol, 4
equivalents) of 5-hexynoic acid were dissolved in 3 mL of dry THF. The reaction mixture was charged
with N2 and then 0.35 mL of TEA (2.5 mmol, 10 equivalents) were added via syringe. After 2 hours at
room temperature the reaction was heated to 70°C overnight. The catalyst was removed by filtration
through Celite, THF removed in vacuo and the crude product thus obtained dissolved in acetonitrile.
Purification via reverse phase HPLC afforded the pure compound.
Characterization Yield : 81 mg (63%) as brown oil Analytical Data : C26H25NO10 (MW = 511,49) APCI-MS m/z = 512.0 [M+1]+ HPLC : tr = 19.6 min. purity 93 % (254nm) IR (film) : 3500-3280, 2986, 2954, 2876, 1731, 1608, 1576, 1455, 1425, 1197, 1051 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.47 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.97 (m, 2H, CH2-CH2-CH2), 2.29 (ddd, 1H, J = 10.6,
4.6, 2.3 Hz, H-4a), 2.33 (t, 2H, J = 7.2 Hz, HOOC-CH2), 2.36 (dd, 1H, J = 12.2, 8.1 Hz, H-5a), 2.72
(dq, 1H, J = 12.2, 6.8 Hz, H-6), 2.79 (m, 3H, H-4 alpha + =C-CH2), 2.92 (dd, 1H, J = 17.5, 4.6 Hz, H-4
beta ), 3.58 (dd, 1H, J = 10.6, 8.1 Hz, H-5), 6.43 (s, 1H, =C-H), 7.21 (d, 1H, J = 7.9 Hz, H-7), 7.58 (d,
1H, J = 7.9 Hz, H-8).
13C NMR (150 MHz, CD3OD) :
Experimental Part
128
δ (ppm) = 17. 80 (CH3), 24.05 (CH2-CH2-CH2), 28.50 (HOOC-CH2), 31.32 (C-4), 34.06 (=C-CH2),
40.33 (C-6), 45.00 (C-4a), 47.85 (C-5a), 70.43 (C-5), 76.88 (C-12a), 100.05 (C-2), 102.87 (HC-C9),
107.87 (C-11a), 116.85 (C-10a), 120.50, 126.62, 130.78 (C-7, C-8, C-9), 143.96 (C-6a), 153.83 (C-
10), 175.04, 176.96 (C-12, CONH2), 182.21, 186.20, 196.43 (COOH, C1, C3, C11).
9-Octa 1’,7’diynyl-4-dedimethylamino doxycycline (25)
132 mg (0.25 mmol) of derivative 6, 0.0289 g (0.025 mmol) of
Tetrakis(triphenylphosphine)palladium(0), 0.0047 g (0.025 mmol) of copper iodide, 0.15 mL (1 mmol, 4
equivalents) of 1,7 octadiyne were dissolved in 3 mL of dry THF. The reaction mixture was charged
with N2 and then 0.35 mL of TEA (2.5 mmol, 10 equivalents) were added via syringe. After 2 hours at
room temperature the reaction was completed. The catalyst was removed by filtration through Celite,
THF removed in vacuo and the crude product thus obtained dissolved in acetonitrile. Purification via
reverse phase HPLC afforded the pure compound.
Characterization Yield : 64 mg (51%) Analytical Data : C28H27NO8 (MW = 505,53) APCI-MS m/z = 506.0 [M+1]+ HPLC : tr = 21.3 min. purity > 98 % (254nm) IR (film) : 3550-3200, 3290, 2982, 2938, 1863, 2229, 2114, 1661, 1600, 1554, 1425, 1274,
1240 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.50 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.67-1.77 (m, 4H, H at C4’ and C5’), 2.19.2.22 (s, 1H,
H at C8’), 2.22-2.28 (m, 2H, H at C6’), 2.34 (ddd, 1H, J = 10.8, 5.4, 2.3 Hz, H-4a), 2.37 (dd, 1H, J =
12.5, 8.9 Hz, H-5a), 2.44-2.51 (m, 2H, H at C3’), 2.69 (dq, 1H, J = 13.0, 6.4 Hz, H-6), 2.92 (dd, 1H, J =
18.5, 2.3 Hz, H-4 alpha ), 3.04 (dd, 1H, J = 18.7, 5.1 Hz, 4-H beta), 3.63 (dd, 1H, J = 10.6, 8.3 Hz, H-
5), 6.87 (d, 1H, J = 7.9 Hz, H-7), 7.48 (d, 1H, J = 7.9 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.21 (CH3), 18.59, 19.77 (C-4’, C-6’), 28.78 (C-3’, C-5’), 31.24 (C-4), 40.01 (C-6), 44.93 (C-
4a), 47.85 (C-5a), 69.65, 69.94 (C-8’, C-5), 75.90, 76.53 (C-12a, C-1’), 84.77 (C-7’), 95.62 (C-2’),
Experimental Part
129
99.61 (C-2), 108.00, 113.22, 116.54, 116.94 (4 Aromatic C), 140.39 (C-8), 149.01 (C-6a), 163.69 (C-
10), 175.06, 175.40 (C-12, CONH2), 195.31, 196.35 (C1, C3, C11).
9-(6-(1,3-dioxoisoindolin-2-yl)hex-1-ynyl)-4-dedimethylamino doxycycline (26)
132 mg (0.25 mmol) of derivative 6, 0.0289 g (0.025 mmol) of
Tetrakis(triphenylphosphine)palladium(0), 0.0047 g (0.025 mmol) of copper iodide, 0.227 grams (1
mmol, 4 equivalents) of 6-phtalimido-1-hexyne were dissolved in 3 mL of dry THF. The reaction
mixture was charged with N2 and then 0.35 mL of TEA (2.5 mmol, 10 equivalents) were added via
syringe. After 2 hours at room temperature the reaction was complete. The catalyst was removed by
filtration through Celite, THF removed in vacuo and the crude product thus obtained dissolved in
acetonitrile. Purification via reverse phase HPLC afforded the pure compound.
Characterization Yield : 11 mg (70%) as greenish powder Analytical Data : C34H30N2O10 (MW = 626,63) APCI-MS m/z = 627.2 [M+1]+ HPLC : tr = 21.4 min. purity > 97 % (254nm) IR (film) : 3500-3200, 2981, 2943, 2872, 1770, 1712, 1641, 1604, 1556, 1425, 1398, 1038
cm-1
1H NMR (600 MHz, CDCl3) :
δ (ppm) = 1.60 (d, 3H, J = 6.2 Hz, CH3 at C-6), 1.69 (dt, 2H, J = 14.9, 7.5, H at C4’), 1.90 (dt, 2H, J =
14.9, 7.5, H at C5’), 2.54 (t, 2H, J = 7.0, H at C3’), 2.66-2.88 (m, 5H, H4,H4a,H5a,H6), 3.73-3.78 (m,
3H, H-5 and H at C6’), 5.88 (br s, 1H, CONH2), 6.86 (d, 1H, J = 7.9 Hz, H-7), 7.53 (d, 1H, J = 7.9 Hz,
H-8), 7.69-7.72 and 7.82-7.86 (2 m, 2x2H, phtalimido H), 9.10 (br s, 1H, CONH2), 12.33 (s, 1H, OH),
14.94 (s, 1H, OH), 18.01 (s, 1H, OH).
Experimental Part
130
9-Cyano-4-dedimethylamino doxycycline (28)
100 mg (0.19 mmol) of derivative 6, 22 mg (0.019 mmol) of Tetrakis(triphenylphosphine)palladium(0),
0.0036 g (0.019 mmol) of copper iodide, 0.0247 grams (1 mmol, 4 equivalents) of potassium cyanide
were dissolved in 4 mL of dry THF. The reaction mixture was charged with N2 and then 0.35 mL of
TEA (2.5 mmol, 10 equivalents) were added via syringe. After 1 hour at reflux the reaction was
complete. The catalyst was removed by filtration through Celite, THF removed in vacuo and the crude
product thus obtained dissolved in acetonitrile. Purification via reverse phase HPLC afforded the pure
compound.
Characterization Yield : 65 mg (80%) as brown-yellow powder Analytical Data : C21H18N2O8 (MW = 426,39) APCI-MS m/z = 427.0 [M+1]+ HPLC : tr = 16.9 min. purity > 99 % (254nm) IR (film) : 3530-3230, 2974, 2874, 2230, 1644, 1610, 1568, 1435, 1119 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.35 (ddd, 1H, J = 10.9, 5.5, 1.5 Hz, H-4a), 2.43 (dd,
1H, J = 12.3, 8.1 Hz, H-5a), 2.80 (dq, 1H, J = 12.8, 6.3 Hz, H-6), 2.92 (dd, 1H, J = 18.5, 1.5 Hz, H-4
alpha ), 3.04 (dd, 1H, J = 18.5, 5.3 Hz, 4-H beta), 3.65 (dd, 1H, J = 10.6, 7.9 Hz, H-5), 7.09 (d, 1H, J =
7.9 Hz, H-7), 7.80 (d, 1H, J = 7.9 Hz, H-8).
13C NMR (150 MHz, CD3OD) :
δ (ppm) = 16.16 (CH3), 31.33 (C-4), 40.39 (C-6), 44.91 (C-4a), 47.28 (C-5a), 69.84 (C-5), 76.00 (C-
12a), 99.51 (C-2), 101.09 (C-9), 107.83 (C-11a), 116.25 (CN), 117.72, 117.81 (C-7, C-10a), 140.54
(C-8), 155.27 (C-6a), 164.51 (C-10), 175.06, 177.13 (C-12, CONH2), 194.21, 196.38 (C1, C3, C11).
Experimental Part
131
7-Iodo-9-phenylethynyl-4-dedimethylamino doxycycline (29)
50 mg (0.076 mmol) of derivative 7, 8.8 mg (0.0076 mmol) of
Tetrakis(triphenylphosphine)palladium(0), 2 mg (0.0076 mmol) of copper iodide, 0.008 ml (1
equivalent) of phenylacetylene were dissolved in 5 mL of dry THF. The reaction mixture was charged
with N2 and then 0.11 mL of TEA (10 equivalents) were added via syringe. The solution was irradiated
with 100W microwave for ten minutes. The catalyst was removed by filtration through Celite, THF
removed in vacuo and the crude product thus obtained dissolved in acetonitrile. Purification via
reverse phase HPLC afforded the pure compound.
Characterization Yield : 31 mg (64%) as dark-yellow glas Analytical Data : C28H22NO8 (MW = 627,39) APCI-MS m/z = 628.1 [M+1]+ HPLC : tr = 22.8 min. purity > 95 % (254nm) IR (film) : 3500-3200, 2972, 2936, 1871, 1731, 1636, 1560, 1432, 1411, 1191, 1048 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.25 (d, 3H, J = 7.5 Hz, CH3 at C-6), 2.25 (ddd, 1H, J = 10.8, 5.0, 1.7 Hz, H-4a), 2.49 (dd,
1H, J = 10.7, 2.0 Hz, H-5a), 2.84 (br d, 1H, J = 18.0 Hz, H-4 alpha ), 2.96 (dd, 1H, J = 18.1, 4-H beta),
3.44 (dd, 1H, J = 10.9, 10.9 Hz, H-5), 3.90 (dq, 1H, J = 7.2, 1.8 Hz, H-6), 7.35-7.39 (m, 3H,
Aromatics), 7.49-7.54 (m, 2H, aromatics), 8.10 (s, 1H, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 19.63 (CH3), 31.51 (C-4), 39.60 (C-6), 44.11 (C-4a), 47.83 (C-5a), 68.06 (C-5), 75.85 (C-
12a), 83.39 (C1’ alkyne), 87.48 (C-7), 96.25 (C2’ alkyne), 99.89 (C-2), 114.90 (C-11a), 117.74 (C-9),
124.19 (C-10a), 129.56, 129.83, 132.65 (6 Aromatic C), 150.57, 150.85 (C-8, C-6a), 163.83 (C-10),
174.69, (C-12, CONH2), 195.51, 197.29, 200.18 (C1, C3, C11).
Experimental Part
132
29 HMBC shows the correlation between proton at C-6 (3.9 ppm) and carbons 6a (150 ppm) and 7
(87ppm). If the cross-coupling reaction had happened at position 7, we would have a correlation with a
carbon at circa 116 ppm.
Experimental Part
133
7,9-bis(phenylethynyl)-4-dedimethylamino doxycycline (30)
50 mg (0.076 mmol) of derivative 7, 8.8 mg (0.0076 mmol) of
Tetrakis(triphenylphosphine)palladium(0), 2 mg (0.0076 mmol) of copper iodide, 0.024 ml (3
equivalent) of phenylacetylene were dissolved in 5 mL of dry THF. The reaction mixture was charged
with N2 and then 0.11 mL of TEA (10 equivalents) were added via syringe. The solution was irradiated
with 100W microwave for ten minutes. The catalyst was removed by filtration through Celite, THF
removed in vacuo and the crude product thus obtained dissolved in acetonitrile. Purification via
reverse phase HPLC afforded the pure compound.
Characterization Yield : 19.6 mg (43%) as yellow-orange glas Analytical Data : C36H27NO8 (MW = 601,62) APCI-MS m/z = 602.1 [M+1]+ HPLC : tr = 23.7 min. purity > 99 % (254nm) IR (film) : 3540-3160, 3058, 2974, 2874, 2211, 1644, 1569, 1490, 1442, 1196, 1055 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.38 (d, 3H, J = 7.2 Hz, CH3 at C-6), 2.26 (ddd, 1H, J = 10.8, 5.0, 1.7 Hz, H-4a), 2.53 (dd,
1H, J = 10.9, 1.8 Hz, H-5a), 2.84 (dd, 1H, J = 18.2, 1.7 Hz, H-4 alpha ), 2.97 (dd, 1H, J = 18.1, 5.0 Hz,
4-H beta), 3.53 (dd, 1H, J = 10.9, 10.9 Hz, H-5), 4.26 (dq, 1H, J = 7.3, 1.8 Hz, H-6), 7.34-7.41 (m, 6H,
Aromatics), 7.50-7.56 (m, 4H, aromatics), 7.82 (s, 1H, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 20.66 (CH3), 26.07 (C-4), 33.13 (C-6), 46.49 (C-4a), 48.31 (C-5a), 68.34 (C-5), 84.15,
86.39, 94.07, 95.87 (C1’, C2’, C1” and C2” alkyne), 100.18 (C-2), 113.13 (C-11a), 115.77 (C-9),
117.10, 124.53 (C-10a, C-7), 129.73, 129.79, 129.92, 132.70, 132.83 (10 Aromatic C), 144.18, 151.49
(C-8, C-6a), 163.71 (C-10), 174.92, 175.00, (C-12, CONH2), 195.71, 200.56 (C1, C3, C11).
Experimental Part
134
General conditions for the Suzuki coupling derivatives: A solution of the respective iodo-4-ddmadoxycycline (6 or 7) (50 mg), PhB(OH)2 (2equiv), Pd(OAc)2
(0.1 equiv) and Na2CO3 in a mixture of DMF and water was irradiated to 80°C with microwavaves for
10 minutes. After consumption of the iodo derivative the mixture was filtered through a pad of Celite™
and purified by preparative HPLC to give pure compounds.
9-Phenyl-4-dedimethylamino doxycycline (31)
0.1g (0.19 mmol) of derivative 6, 4.3 mg (0.019 mmol) of Pd(OAc)2 and 46.3 mg (0.38 mmol, 2
equivalents) of phenylboronic acid were dissolved in 3 mL DMF. To this mixture 61.3 mg of Na2CO3
(3 equivalents) in 1mL water were added, and the vial was irradiated with microwaves for 10 minutes
at 80 °C.The mixture was then diluted with MeOH, filtered through Celite to remove the catalyst, the
solvent removed in vacuo and the raw product is purified through RP-HPLC.
Characterization Yield : 37 mg (74%) as dark brown glas Analytical Data : C26H23NO8 (MW =477,48) APCI-MS m/z = 478.1 [M+1]+ HPLC : tr = 22.1 min. purity > 99 % (254nm) 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.55 (d, 3H, J = 6.7 Hz, CH3 at C-6), 2.36 (ddd, 1H, J = 10.9, 5.3, 2.3 Hz, H-4a), 2.42 (dd,
1H, J = 12.5, 7.9 Hz, H-5a), 2.75 (dq, 1H, J = 13.0, 6.6 Hz, H-6), 2.94 (dd, 1H, J = 18.5, 2.2 Hz, H-4
alpha ), 3.05 (dd, 1H, J = 18.5, 5.3 Hz, 4-H beta), 3.66 (dd, 1H, J = 10.7, 8.1 Hz, H-5), 7.02 (d, 1H, J =
7.9 Hz, H-7), 7.31 (dd, 1H, J = 7.3, 7.3, H at C4’), 7.39 (t, 2H, J = 7.6, H at C2’ and C6’), 7.52-7.58 (m,
3H, H-8, H at C3’ and C5’).
Experimental Part
135
9-(p-carboxyphenyl)-4-dedimethylamino doxycycline (32)
0.1g (0.19 mmol) of derivative 6, 4.3 mg (0.019 mmol) of Pd(OAc)2 and 63 mg (0.38 mmol, 2
equivalents) of 4-carboxy-phenylboronic acid were dissolved in 3 mL DMF. To this mixture 61.3 mg of
Na2CO3 in 1mL water were added, and the mixture was irradiated with microwaves for 10 minutes at
80 °C.The mixture is then diluted with MeOH, filtered through Celite to remove the catalyst, the solvent
removed in vacuo and the raw product is purified through RP-HPLC.
Characterization Yield : 64 mg (65%) as light-brown oil Analytical Data : C27H23NO10 (MW =521,49) APCI-MS m/z = 522.1 [M+1]+ HPLC : tr = 20.1 min. purity 94 % (254nm) IR (film) : 3540-3200, 2974, 2936, 2876, 1714, 1650, 1605, 1555, 1428, 1402, 1277, 1242,
1184, 1130, 1051 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.36 (ddd, 1H, J = 10.8, 5.4, 2.2 Hz, H-4a), 2.42 (dd,
1H, J = 12.5, 7.9 Hz, H-5a), 2.74 (dq, 1H, J = 13.0, 6.8 Hz, H-6), 2.94 (dd, 1H, J = 18.6, 2.1 Hz, H-4
alpha ), 3.05 (dd, 1H, J = 18.6, 5.4 Hz, 4-H beta), 3.66 (dd, 1H, J = 10.7, 8.1 Hz, H-5), 7.03 (d, 1H, J =
8.0 Hz, H-7), 7.58 (d, 1H, J = 8.0 Hz, H-8), 7.68 (d, 2H, J = 8.3 Hz, H at C2’ and C6’), 8.05 (d, 2H, J =
8.3 Hz, H at C3’ and C5’).
13C NMR (150 MHz, CD3OD) :
δ (ppm) = 16.24 (CH3), 31.26 (C-4), 40.11 (C-6), 44.93 (C-4a), 47.98 (C-5a), 69.90 (C-5), 75.91 (C-
12a), 99.62 (C-2), 108.19 (C-11a), 116.93, 117.37 (C-10a, C-7), 128.29, 128.69, 130.34, 130.40,
130.49, 131.43 (6 Aromatic C), 138.15 (C-8), 143.21 (C-1’), 149.94 (C-6a), 160.62 (C-10), 169.79
(COOH), 175.08, 175.27 (C-12, CONH2), 195.80, 196.40 (C1, C3, C11).
Experimental Part
136
7, 9-diphenyl-4-dedimethylamino doxycycline (33)
50 mg (0.076 mmol) of derivative 7, 1.7 mg (0.0076 mmol) of Pd(OAc)2 and 37.1 mg (0.30 mmol, 4
equivalents) of phenylboronic acid were dissolved in 3 mL DMF. To this mixture 24 mg of Na2CO3
(0.23 mmol, 3 equivalents) in 1mL water were added, and the vial was irradiated with microwaves for
10 min at 100 °C.The mixture was then diluted with MeOH, filtered through Celite to remove the
catalyst, the solvent removed in vacuo and the raw product is purified through RP-MPLC.
Characterization Yield : 24 mg (57%) as yellow film Analytical Data : C32H27NO8 (MW =553,57) APCI-MS m/z = 554.2 [M+1]+ HPLC : tr = 23.3 min. purity 95 % (254nm) IR (film) : 3485-3150, 3061, 2982, 1643, 1607, 1557, 1428, 1400, 1280, 1242, 1200, 1130,
1037 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.07 (d, 3H, J = 7.2 Hz, CH3 at C-6), 2.24 (ddd, 1H, J = 10.8, 4.8, 2.0 Hz, H-4a), 2.39 (dd,
1H, J = 10.9, 2.2 Hz, H-5a), 2.89 (br dd, 1H, J = 18.2 Hz, H-4 alpha ), 2.99 (dd, 1H, J = 18.2 Hz, 4-H
beta), 3.68 (dd, 1H, J = 10.9, 10.9 Hz, H-5), 3.81 (dq, 1H, J = 10.9, 7.2 Hz, H-6), 7.28-7.32 (m, 2H H at
C4’ and C4’’), 7.34-7.40 (m, 4H, H at C3’, C5’, C3’’, C5’’), 7.42 (d, 4H, H at C2’, C6’, C2’’, C6’’), 7.51
(d, 1H, J = 7.6 Hz, H-8).
13C NMR (150 MHz, CD3OD) :
δ (ppm) = 21.16 (CH3), 31.18 (C-4), 41.02 (C-6), 44.29 (C-4a), 47.98 (C-5a), 68.42 (C-5), 82.62 (C-
12a), 101.49 (C-2), 108.19 (C-11a), 116.71, 117.35 (C-10a, C-7), 128.48, 129.27, 129.43, 129.67,
130.46, 130.51, 130.93, 134.99, 138.07, 140.86, 141.18 (Aromatic C), 145.26 (C-6a), 160.00 (C-10),
174.84, 178.33 (C-12, CONH2), 196.12, 200.73 (C1, C3, C11).
Experimental Part
137
9-Iodo doxycycline (34)
Two grams of doxycycline (MW=462, 4.33 mmol) were dissolved in 10 mL of trifluoroacetic acid that
was cooled to 0° C (on ice). N-iodosuccinimide (1.1 equivalents, 1.07 grams) was added to the
reaction in three portions every 15 minutes. After 2 hours the reaction was complete, the mixture was
dripped slowly in 500 mL of ice-cold ether. The precipitate thus obtained was filtrated, washed several
times with cold ether, collected and dried in vacuum overnight to yield 9-iodo doxycycline without
further purification.
Characterization Yield : 2.23 g (90.2%) Analytical Data : C22H23IN2O8 (MW = 570,34) APCI-MS m/z = 571.2 [M+1]+ HPLC : tr = 18.5 min. purity > 95 % (254nm) IR (film) : 3450-3200, 3061, 2975, 2877, 1707, 1671, 1616, 1574, 1416, 1201, 1136, 1043 cm-1
Further analytical data described in literature (Nelson et al. J. Org. Chem., 68 (15), 5838 -5851, 2003)
9-(6-(1,3-Dioxoisoindolin-2-yl)hex-1-ynyl) doxycycline (35)
200 mg (0.35 mmol) of derivative 34, 40.4 mg (0.035 mmol) of
Tetrakis(triphenylphosphine)palladium(0), 6.6 mg (0.035 mmol) of copper iodide, 318 mg (4
equivalents) of 6-phtalimido-1-hexyne were dissolved in 5 mL of dry THF. The reaction mixture was
charged with N2 and then 0.49 mL of TEA (10 equivalents) were added via syringe. The solution was
stirred at 40°C for 1.5 hours. The mixture was diluted with MeOH/HCl (10 + 1 mL) and the catalyst
Experimental Part
138
removed by filtration through Celite. Solvents were removed in vacuo and the crude product obtained
used without further purifications.
Characterization Yield : 214 mg (91%, crude) as dark brown glas Analytical Data : C36H35N3O10 (MW = 669,69) APCI-MS m/z = 654.1 [M+1]+ - 17 HPLC : tr = 19.6 min. purity 90 % (254nm) IR (film) : 3500-3100, 2975, 2944, 2871, 2232, 1770, 1713, 1680, 1605, 1555, 1425, 1397,
1200, 1132, 1064 cm-1
1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.53 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.65 and 1.90 (2 m, each 2H, central CH2CH2 side
chain), 2.52 (t, 2H, J = 6.8 Hz, CH2-≡), 2.56 (dd, 1H, J = 12.0, 8.2 Hz, H-5a), 2.74 (dq, 1H, J = 12.0,
6.2 Hz, H-6), 2.81 (d, 1H, J = 11.3, H-4a), 2.96 (s, 6H, N(CH3)2), 3.56 (dd, 1H, J = 11.5, 8.3 Hz, H-5),
3.74 (t, 2H, J = 6.9 Hz, CH2-N), 4.40 (s, 1H, H-4), 6.89 (d, 1H, J = 8.0 Hz, H-7), 7.50 (d, 1H, J = 8.0
Hz, H-8), 7.78 (dd, 2H, J = 5.6, 2.8 Hz, H at C-4’ and 5’ phtalimido), 7.83 (dd, 2H, J = 5.6, 2.8 Hz, H at
C-3’ and 6’ phtalimido).
Tert-butyl 3-((5R,6S,7S,10aS)-9-carbamoyl-7-(dimethylamino)-1,6,8,10a,11-pentahydroxy-5-methyl-10,12-dioxo-5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-
yl)prop-2-ynylcarbamate (37)
50 mg (0.087 mmol) of 9-iodo doxycycline 34, 10 mg (0.0087 mmol) of
Tetrakis(triphenylphosphine)palladium(0), 2 mg (0.009 mmol) of copper iodide, 54.3 mg (4
equivalents) of N-Boc propargylamine were dissolved in 2 mL of dry THF. The reaction mixture was
charged with N2 and then 0.12 mL of TEA (10 equivalents) were added via syringe. The solution was
stirred at 40°C for 1.5 hours. The mixture was diluted with MeOH/HCl (10 + 1 mL) and the catalyst
removed by filtration through Celite. Solvents were removed in vacuo and the crude product obtained
purified with reverse phase HPLC.
Experimental Part
139
Characterization Yield : 22 mg (42%) as light yellow glas Analytical Data : C30H35N3O10 (MW = 597,63) APCI-MS m/z = 598.3 [M+1]+ HPLC : tr = 17.8 min. purity > 99 % (254nm) IR (film) : 3540-3180, 3071, 2975, 2879, 2130, 1672, 1607, 1555, 1426, 1201, 1135 cm-1 1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.47 (s, 9H, 3 CH3 Boc), 1.53 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.53-2.64 (m, 2H, H-5a and
H-6), 2.81 (d, 1H, J = 11.3, H-4a), 2.94 (s, 6H, N(CH3)2), 3.56 (m, 1H, H-5), 4.08 (s, 2H, CH2-≡), 4.40
(s, 1H, H-4), 6.93 (d, 1H, J = 8.0 Hz, H-7), 7.57 (d, 1H, J = 8.0 Hz, H-8).
After HPLC, two sets of signals appear in the aromatic region, due to benzofuran ring formation :
6.94 and 7.00 ( 2 d, each 1H, J = 7.9 HZ, H-7), 7.58 and 7.65 ( 2d, each 1H, J = 7.9 Hz, H-8), 7.98 (s,
1H, CH=).
9-Nitro doxycycline (38)
Doxycycline (10 grams, 21.6 mmol) was dissolved in 40 mL of concentrated H2S04, cooled to 0°C and
NaN03 (2.87 g, 33.8 mmol) was added over 10 min. The reaction mixture was stirred an additional 3 h
and then diluted with 30 mL of methanol. The solution was dripped into ice-cooled, stirred ether (2 L),
and the mixture was filtered. The precipitate was washed well with ether, vacuum dried and used
without further purification.
Characterization Yield : 7.45 grams (70%, crude) as pale yellow solid Analytical Data : C22H23N3O10 (MW = 489,44) APCI-MS m/z = 490.1 [M+1]+ HPLC : tr = 15.3 min. IR (film) : 3470-3150, 3082, 2974, 2878, 1669, 1621, 1584, 1524, 1457, 1427, 1346, 1202,
1170, 1042, 853 cm-1
Experimental Part
140
Further analytical data described in literature (Barden et al. J. Med. Chem. 1994, 37, 3205-3211).
9-Amino doxycycline (39)
Crude 9-nitro doxycycline 38 (1 gram, 2.04 mmol) was dissolved in 25 mL of methanol and poured into
a 500 mL Paar hydrogenation bottle. 10% Pd on charcoal (0.1 g) and 2.5 mL of concentrated HCl
were added, the system was charged with 50 psi of H2, and the bottle was stirred at 30°C overnight.
After filtration of the catalyst through Celite, the solution was diluted to 50 mL with methanol containing
HCl and rapidly dripped into cold stirred ether (1 L) to give a light tan powder. Portions were purified
by preparative HPLC as needed.
Characterization Yield : 850 mg (90%, crude) Analytical Data : C22H25N3O8 (MW = 459,46) APCI-MS m/z = 460.1 [M+1]+ HPLC : tr = 4.4 min. purity > 95 % (254nm) IR (film) : 3470-3260, 3090, 2955, 2876, 1733, 1670, 1615, 1558, 1507, 1244, 1134 cm-1 Further analytical data described in literature (Barden et al. J. Med. Chem. 1994, 37, 3205-3211).
Experimental Part
141
General procedure for the synthesis of Boc-amino acid symmetric anhydrides: 1 mmol of N-Boc amino acid was dissolved in 5 ml of dichloromethane and cooled to 0°C in an ice
bath. To this stirring solution 0.5 mmol of DCC (dicyclohexyl carbodiimide) dissolved in 1 mL of DCM
were added dropwise and the reaction proceeded for 30 minutes. After 1h storage at -20°C, the
precipitate formed was filtered out, and the solvent evaporated at reduced pressure. The crude
symmetric anhydride thus obtained was used for the acylation step without further purification.
9-(N-Boc-glycylamino) doxycycline (40)
To a solution of 100 mg (0.217 mmol) of amino derivative 39 in 5 mL of DMF, containing 2 equivalents
of NaHCO3 (0.43 mmol, 36.6 mg), 3 equivalents of N-Boc glycine symmetric anhydride (0.66 mmol)
obtained with the described general procedure are slowly dropped in. The reaction proceeds to
completeness at room temperature whithin 3 hours, as confirmed by LC-MS analysis. The solvent is
then removed in vacuo and the raw product is purified through RP-HPLC.
Characterization Yield : 90 mg (67%) as black film Analytical Data : C29H36N4O11 (MW = 616,63) APCI-MS m/z = 617.4 [M+1]+ HPLC : tr = 16.7 min. purity 89 % (254nm) IR (film) : 3520-3130, 3088, 2979, 2876, 1675, 1614, 1536, 1244, 1200, 1176, 1135, 1051
cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.49 (s, 9H, 3 CH3 Boc), 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.56 (dd, 1H, J = 12.0, 8.3 Hz,
H-5a), 2.75 (dq, 1H, J = 13.2, 6.6 Hz, H-6), 2.82 (d, 1H, J = 11.3, H-4a), 2.96 (s, 6H, N(CH3)2), 3.56
(dd, 1H, J = 11.3, 8.3 Hz, H-5), 3.90 (s, 2H, CH2 gly), 4.41 (s, 1H, H-4), 6.95 (d, 1H, J = 8.3 Hz, H-7),
8.34 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (150 MHz, CD3OD) :
δ (ppm) = 16.12 (CH3), 28.71 (CH3 Boc), 39.76 (C-4a), 42.96 (C-6), 43.03 (CH2 gly), 45.53 (N(CH3)2),
48.01 (C-5a), 67.08 (C-4), 69.99 (C-5), 74.66 (C-12a), 81.04 (C(CH3)3), 101.43 (C-2), 108.52 (C-11a),
Experimental Part
142
116.28, 117.20 (C-7,C-10a), 126.52, 128.33 (C-9, C-8), 144.00 (C-6a), 155.43 (C-10), 158.61
(NHCOOC(CH3)3), 170.86, 173.03, 174.08 (C-12, CONH2, CONH), 195.58 (C1, C3, C11).
9-( 5’-Boc-amino)pentinoylamino Doxycycline (41)
To a solution of 100 mg (0.217 mmol) of amino derivative 39 in 5 mL of DMF, containing 2 equivalents
of NaHCO3 (0.43 mmol, 36.6 mg), 2 equivalents of N-Boc-5-aminopentanoic acid symmetric anhydride
(0.44 mmol) obtained with the described general procedure are slowly dropped in. The reaction
proceeds to completeness at room temperature whithin 3 hours, as confirmed by LC-MS analysis. The
solvent is then removed in vacuo and the raw product is purified through RP-HPLC.
Characterization Yield : 114 mg (79%) as greenish powder Analytical Data : C32H42N4O11 (MW = 658,71) APCI-MS m/z = 659.3 [M+1]+ HPLC : tr = 17.3 min. purity > 98 % (254nm) IR (film) : 3500-3100, 3060, 2977, 2877, 1672, 1615, 1525, 1243, 1200, 1133, 1141 cm-1
1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.43 (s, 9H, 3 CH3 t-Bu), 1.55 (d, 3H, J = 6.2 Hz, CH3 at C-6), 1.68-1.81 (m, 4H, central
CH2CH2 side chain), 2.48 and 2.55 (2 t, each 1H, J = 7.1 Hz, diastereotopic CH2-CONH), 2.58 (dd, 1H,
J = 12.4, 8.4 Hz, H-5a), 2.75 (dq, 1H, J = 13.2, 6.2 Hz, H-6), 2.82 (d, 1H, J = 11.4, H-4a), 2.95 (s, 6H,
N(CH3)2), 2.98 and 3.09 (2 t, each 1H, J = 6.8 Hz, diastereotopic Boc-NH-CH2), 3.57 (dd, 1H, J = 11.4,
8.4 Hz, H-5), 4.40 (s, 1H, H-4), 6.95 (d, 1H, J = 8.4 Hz, H-7), 8.15 (d, 1H, J = 8.4 Hz, H-8).
9-Glycylamino doxycycline (42)
Experimental Part
143
50 mg (0.08 mmol) of Boc-amino derivative 40 are dissolved in 5 mL of 50% TFA in DCM and stirred
for 1 hour at room temperature. The solvent (DCM) is then removed in vacuo and the raw product is
diluted with methanol and purified through RP-HPLC.
Characterization Yield : 12 mg (29%) as yellow film Analytical Data : C24H28N4O9 (MW = 516,51) APCI-MS m/z = 517.4 [M+1]+ HPLC : tr = 3.0 min. purity 83 % (254nm) IR (film) : 3650-3200, 3065, 2978, 2879, 1713, 1682, 1614, 1538, 1434, 1204, 1133, 1054,
839, 801, 723 cm-1
Further analytical data described in literature (Barden et al. J. Med. Chem. 1994, 37, 3205-3211).
9-(5’-Amino-pentanamido) doxycycline (43)
50 mg (0.076 mmol) of Boc-amino derivative 41 were dissolved in 5 mL of 50% TFA in DCM and
stirred for 1 hour at room temperature. The solvent (DCM) was removed in vacuo and the raw product
diluted with methanol and purified through RP-HPLC.
Characterization Yield : 39 mg (92%) as yellow film Analytical Data : C27H34N4O9 (MW = 558,59) APCI-MS m/z = 559.3 [M+1]+ HPLC : tr = 10.1 min. purity > 99 % (254nm) IR (film) : 3500-3200, 3064, 2972, 2878, 1673, 1529, 1427, 1241, 1200, 1178, 1132, 1040
cm-1
1H NMR (360 MHz, CD3OD) :
Experimental Part
144
δ (ppm) = 1.55 (d, 3H, J = 6.4 Hz, CH3 at C-6), 1.63-1.84 (m, 4H, central CH2CH2 side chain), 2.54 (t,
2H, J = 6.6 Hz, CH2-CONH), 2.58 (dd, 1H, J = 12.4, 8.4 Hz, H-5a), 2.76 (dq, 1H, J = 12.6, 6.2 Hz, H-
6), 2.82 (d, 1H, J = 11.3, H-4a), 2.92-3.02 (m, 8H, N(CH3)2 + NH-CH2), 3.57 (dd, 1H, J = 11.5, 8.3 Hz,
H-5), 4.42 (s, 1H, H-4), 6.95 (d, 1H, J = 8.4 Hz, H-7), 8.15 (d, 1H, J = 8.4 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.20 (CH3), 23.39 (CH2-CH2-CO), 28.04, 28.15 (C-4a, CH2-CH2-NH2), 36.64 (CH2-CO).
39.92 (C-6), 40.52 (CH2-NH2), 43.15 (N(CH3)2), 48.11 (C-5a), 67.18 (C-4), 70.11 (C-5), 74.73 (C-12a),
96.23 (C-2), 108.59 (C-11a), 117.23, 119.17 (C-7,C-10a), 126.67 (C-9), 130.39 (C-8), 144.84 (C-6a),
154.27 (C-10), 173.15, 174.28 (C-12, CONH2, CONH), 195.73 (C1, C3, C11).
9-(5’-Acetamido-pentanamido) doxycycline (43a)
10 mg (0.018 mmol) of derivative 43 were dissolved in 2 mL of DMF. To this solution 2 equivalents of
NaHCO3 (3 mg) were added and 1.5 equivalents of acetic anhydride (0.0025 mL) were dropwise
injected. The mixture was stirred at room temperature and analyzed by LC-MS after 1 hour. The
solution was diluted with MeOH (2 mL) and purified through RP-HPLC.
Characterization Yield : 9 mg (83%) as dark yellow film Analytical Data : C29H36N4O10 (MW = 600,63) APCI-MS m/z = 601.3 [M+1]+ HPLC : tr = 14.0 min. purity > 99 % (254nm) 1H NMR (360 MHz, CD3OD) :
δ (ppm) = 1.50-1.68 (m, 5H, CH3 at C-6 and CH2-CH2-NH), 1.68-1.79 (m, 2H, CH2CH2CO), 1.93 (s,
3H, CH3 acetyl), 2.54 (t, 2H, J = 6.6 Hz, CH2-CONH), 2.58 (m, 1H, H-5a), 2.69-2.87 (m, 2H, H-6 and
H-4a), 2.95 (br s, 6H, N(CH3)2), 3.21 (t, 2H, J = 6.9 Hz, NH-CH2), 3.56 (br s, 1H, H-5), 4.41 (s, 1H, H-
4), 6.95 (d, 1H, J = 7.9 Hz, H-7), 8.15 (d, 1H, J = 7.9 Hz, H-8).
(weak and unsharp signals due to low substance concentration).
Experimental Part
145
9-(8’-Boc-amino-octanamido) doxycycline (44)
To a solution of 200 mg (0.435 mmol) of amino derivative 39 in 7 mL of DMF, containing 2 equivalents
of NaHCO3 (0.73 mmol, 87 mg), 2 equivalents of N-Boc-8-aminooctanoic acid symmetric anhydride
(0.87 mmol) obtained with the described general procedure and solubilized in DCM/DMF (1+1 mL)
were slowly dropped in. The reaction proceeded to completeness at room temperature whithin 5
hours, as confirmed by LC-MS analysis. The solvent is then removed in vacuo and a part of the raw
product is purified through RP-HPLC for chemical characterization analysis.
Characterization Yield : 198 mg (65% crude) Analytical Data : C35H48N4O11 (MW = 700,79) APCI-MS m/z = 701.5 [M+1]+ HPLC : tr = 19.2 min. purity > 97 % (254nm) IR (film) : 3470-3150, 2977, 2932, 2859, 1672, 1615, 1524, 1243, 1202, 1177, 1134, 1043
cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.30-1.50 (m, 17H, Boc and central CH2s side chain), 1.55 (d, 3H, J = 6.8 Hz, CH3 at C-6),
1.71 (tt, 2H, J = 7.4, 7.3 Hz, CH2-CH2-CO), 2.45 (t, 2H, J = 7.6 Hz, CH2-CONH), 2.57 (dd, 1H, J =
12.3, 8.5 Hz, H-5a), 2.76 (dq, 1H, J = 13.2, 6.4 Hz, H-6), 2.81 (d, 1H, J = 11.3, H-4a), 2.92 (br s, 6H,
N(CH3)2), 3.02 (t, 2H, J = 6.8 Hz, NH-CH2), 3.56 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 4.41 (s, 1H, H-4), 6.95
(d, 1H, J = 8.6 Hz, H-7), 8.15 (d, 1H, J = 8.6 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.13 (CH3), 26.84, 27.72, 28.80, 29.91, 30.07, 30.12, 30.20, 30.92, 34.92 (C-4a), 37.68
(CH2-CO). 39.81 (C-6), 41.33 (CH2-NH-Boc), 43.00 (N(CH3)2), 48.03 (C-5a), 70.00 (C-4, C-5), 74.67
(C-12a), 79.81 (C(CH3)3), 96.29 (C-2), 101.41, 108.59 (C-11a), 116.14, 117.23 (C-7,C-10a), 126.68
(C-9), 130.23 (C-8), 144.47 (C-6a), 154.17 (C-10), 158.60 (COOC(CH3)3), 174.09, 175.07 (C-12,
CONH2, CONH), 191.77, 195.66 (C1, C3, C11).
Experimental Part
146
9-(8’-Amino-octanamido) doxycycline (46)
50 mg (0.071 mmol) of Boc-amino derivative 44 were dissolved in 5 mL of 50% TFA in DCM and
stirred for 1 hour at room temperature. The solvent (DCM) was removed in vacuo and the raw product
diluted with methanol and purified through RP-HPLC.
Characterization Yield : 39 mg (90%) as light yellow powder Analytical Data : C30H40N4O9 (MW = 600,67) APCI-MS m/z = 601.8 [M+1]+ HPLC : tr = 13.1 min. purity > 97 % (254nm) IR (film) : 3500-3200, 3046, 2981, 2937, 2864, 1673, 1614, 1524, 1427, 1241, 1201, 1181,
1134, 1044 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.38-1.48 (m, 6H, central CH2s side chain), 1.55 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.62-1.70
and 1.70-1.77 (2 m, each 2H, CH2-CH2-CO and CH2-CH2-NH2), 2.47 (t, 2H, J = 7.6 Hz, CH2-CONH),
2.58 (dd, 1H, J = 12.1, 8.3 Hz, H-5a), 2.76 (dq, 1H, J = 13.2, 6.4 Hz, H-6), 2.81 (d, 1H, J = 10.6, H-4a),
2.92 (t, 2H, 7.7 Hz, NH-CH2), 2.95 (br s, 6H, N(CH3)2), 3.57 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 4.41 (s,
1H, H-4), 6.95 (d, 1H, J = 8.3 Hz, H-7), 8.15 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.12 (CH3), 26.63, 27.24, 28.52, 29.88, 29.94, 37.56 (CH2-CONH), 39.81 (C-6), 40.74
(CH2-NH2), 43.05 (N(CH3)2), 48.02 (C-5a), 67.20 (C-5), 70.03 (C-12a), 74.72 (C-4), 96.50 (C-2),
108.51 (C-11a), 116.14, 116.91 (C-7,C-10a), 126.64 (C-9), 130.17 (C-8), 144.54 (C-6a), 154.14 (C-
10), 173.13, 174.03, 174.93 (C-12, CONH2, CONH), 188.10, 195.65 (C1, C3, C11).
Experimental Part
147
9-(11’-Boc-amino-undecanamido) doxycycline (45)
To a solution of 200 mg (0.435 mmol) of amino derivative 39 in 7 mL of DMF, containing 2 equivalents
of NaHCO3 (0.73 mmol, 87 mg), 2 equivalents of N-Boc-11-aminoundecanoic acid symmetric
anhydride (0.87 mmol) obtained with the described general procedure and solubilized in DCM/DMF
(1+1 mL) were slowly dropped in. The reaction proceeded to completeness at room temperature
whithin 5 hours, as confirmed by LC-MS analysis. The solvent is then removed in vacuo and a part of
the raw product is purified through RP-HPLC for chemical characterization analysis.
Characterization Yield : 181 mg (56%, crude) as yellow powder Analytical Data : C38H54N4O11 (MW = 742,87) APCI-MS m/z = 743.8 [M+1]+ HPLC : tr = 20.8 min. purity > 95 % (254nm) IR (film) : 3500-3200, 2974, 2928, 2855, 1672, 1652, 1609, 1523, 1244, 1177,
1134, 1059 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.26-1.49 (m, 25H, Boc and central CH2s side chain), 1.55 (d, 3H, J = 6.4 Hz, CH3 at C-6),
1.71 (m, 2H, CH2-CH2-CO), 2.46 (t, 2H, J = 7.4 Hz, CH2-CONH), 2.57 (dd, 1H, J = 12.5, 8.3 Hz, H-5a),
2.75 (dq, 1H, J = 13.2, 6.4 Hz, H-6), 2.81 (d, 1H, J = 11.3, H-4a), 2.95 (br s, 6H, N(CH3)2), 3.01 (t, 2H,
J = 7.0 Hz, NH-CH2), 3.56 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 4.41 (s, 1H, H-4), 6.94 (d, 1H, J = 8.3 Hz, H-
7), 8.15 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.15 (CH3), 26.90, 27.42, 27.85, 30.38, 30.42, 30.51, 30.60, 30.98, 34.75 (C-4a), 37.73
(CH2-CO). 39.80 (C-6), 41.38 (CH2-NH-Boc), 43.03 (N(CH3)2), 48.09 (C-5a), 67.17, 70.00 (C-4, C-5),
74.68 (C-12a), 79.78 (C(CH3)3), 96.35 (C-2), 108.51 (C-11a), 116.13, 116.89 (C-7,C-10a), 126.69,
130.17 (C-9, C-8), 144.43 (C-6a), 154.14 (C-10), 158.57 (COOC(CH3)3), 172.97, 174.08, 175.07 (C-
12, CONH2, CONH), 195.48, 195.63 (C1, C3, C11).
Experimental Part
148
9-(11’-Amino-undecanamido) doxycycline (47)
50 mg (0.067 mmol) of Boc-amino derivative 45 were dissolved in 5 mL of 50% TFA in DCM and
stirred for 1 hour at room temperature. The solvent (DCM) was removed in vacuo and the raw product
diluted with methanol and purified through RP-HPLC.
Characterization Yield : 37 mg (86%) as yellow powder Analytical Data : C33H46N4O9 (MW = 642,76) APCI-MS m/z = 643.7 [M+1]+ HPLC : tr = 15.8 min. purity 95 % (254nm) IR (film) : 3500-3200, 3099, 2981, 2929, 2857, 1678, 1611, 1523, 1432, 1202, 1181,
1135, 835, 799, 722 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.30-1.43 (m, 12H, central CH2s side chain), 1.55 (d, 3H, J = 6.7 Hz, CH3 at C-6), 1.61-1.68
and 1.68-1.74 (2 m, each 2H, CH2-CH2-CO and CH2-CH2-NH2), 2.46 (t, 2H, J = 7.4 Hz, CH2-CONH2),
2.57 (dd, 1H, J = 12.3, 8.1 Hz, H-5a), 2.75 (dq, 1H, J = 12.8, 6.4 Hz, H-6), 2.80 (d, 1H, J = 11.6, H-4a),
2.91 (t, 2H, J = 7.5 Hz, NH2-CH2), 2.95 (br s, 6H, N(CH3)2), 3.57 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 4.40
(s, 1H, H-4), 6.94 (d, 1H, J = 8.3 Hz, H-7), 8.15 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.13 (CH3), 25.98, 26.87, 27.42, 28.58, 30.13, 30.16, 30.25, 30.42, 34.77 (C-4a), 37.72
(CH2-CO). 39.81 (C-6), 40.72 (CH2-NH2), 43.06 (N(CH3)2), 48.02 (C-5a), 70.05, 70.86 (C-4, C-5),
74.77 (C-12a), 96.35 (C-2), 108.50 (C-11a), 116.63, 116.90 (C-7,C-10a), 126.66, 130.16 (C-9, C-8),
144.50 (C-6a), 154.14 (C-10), 173.17, 173.99, 175.05 (C-12, CONH2, CONH), 195.48, 195.63 (C1,
C3, C11).
Experimental Part
149
9-(Boc-11’-amino-3’,6’,9’-trioxaundecanamido) doxycycline (48)
To a solution of 50 mg (0.108 mmol) of amino derivative 39 in 3 mL of DMF, containing 2 equivalents
of NaHCO3 (0.22 mmol, 18 mg), 2 equivalents of N-Boc-11-amino-3,’,’-trioxaundecanic acid symmetric
anhydride (0.22 mmol) obtained with the described general procedure are slowly dropped in. The
reaction proceeds to completeness at room temperature whithin 2 hours, as confirmed by LC-MS
analysis. The solvent is then removed in vacuo and the raw product is purified through RP-HPLC.
Characterization Yield : 23 mg (28%) as brown oil Analytical Data : C35H48N4O14 (MW = 748,79) APCI-MS m/z = 749.4 [M+1]+ HPLC : tr = 17.8 min. purity 90 % (254nm) 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.41 (s, 9H, 3 CH3 Boc), 1.53 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.55 (dd, 1H, J = 12.5, 8.3 Hz,
H-5a), 2.73 (dq, 1H, J = 13.6, 6,5 Hz, H-6), 2.83 (d, 1H, J = 11.3, H-4a), 2.97 (s, 6H, N(CH3)2), 3.19 (t,
2H, J = 5.67 Hz, NHCH2CH2O), 3.48 (t, 2H, J = 5.67 Hz, NHCH2CH2O), 3.56 (dd, 1H, J = 11.3, 8.3 Hz,
H-5), 3.58-3.64, 3.65-3.71, 3.74-3.78 and 3.80-3.84 (4 m, 8H, 2 OCH2CH2O), 4.20 (s, 2H, OCH2CO),
4.41 (s, 1H, H-4), 6.95 (d, 1H, J = 8.3 Hz, H-7), 8.37 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (150 MHz, CD3OD) :
δ (ppm) = 16.15 (CH3), 28.77 (CH3 Boc), 35.37 (C-4a), 39.77 (C-6), 41.28 (CH2NHBoc), 42.97
(N(CH3)2), 47.92 (C-5a), 67.12 (C-4), 70.03 (C-5), 71.09, 71.29, 71.57, 71.67, 71.73, 72.37 (MiniPeg
linker), 74.66 (C-12a), 101.40 (C-2), 108.48 (C-11a), 116.38, 116.77 (C-7,C-10a), 126.12, 128.06 (C-
9, C-8), 144.17 (C-6a), 152.96 (C-10), 158.43 (NHCOOC(CH3)3), 170.77, 173.16, 174.07 (C-12,
CONH2, CONH), 195.43 (C1, C3, C11).
Experimental Part
150
9-(11’-Amino-3’,6’,9’-trioxaundecanamido) doxycycline (49)
23 mg (0.031 mmol) of Boc-amino derivative 48 were dissolved in 4 mL of 50% TFA in DCM and
stirred for 1 hour at room temperature. The solvent (DCM) was removed in vacuo and the raw product
diluted with methanol and purified through RP-HPLC.
Characterization Yield : 2 mg (10%) as brownish powder Analytical Data : C30H40N4O12 (MW = 648,67) APCI-MS m/z = 650.4 [M+1]+ HPLC : tr = 12.7 min. purity 90 % (254nm)
Experimental Part
151
Hex-5-ynoic isobutyric anhydride (50)
0.072 mL (0.65 mmol) of 5-hexynoic acid and 0.068 mL (0.065 mmol) of isobutyl acid chloride were
dissolved In 4 mL of dry DCM. To the solution 1.2 equivalents (0.78 mmol, 0.128 mL) of DIPEA were
added dropwise and the reaction stirred at room temperature overnight. The solution was diluted with
50 mL of hexane, washed 3 times with 0.5N HCl (10 mL) dried with MgSO4, filtered and concentrated
under vacuum.
Yield : 112 mg (95%) as light yellow oil Analytical Data : C10H14O3 (MW = 182,22) IR (film) : 3291, 2978, 2940, 2880, 2118, 1814, 1746, 1470, 1026 cm-1
9-(Hex-5-ynamido) doxycycline (51)
100 mg (0.22 mmol) of 9-amino doxycycline (39) were dissolved in 5 mL of dry DMF and 2 equivalents
(0.43 mmol, 36.6 mg) of NaHCO3 were added. To the mixture were dropped 3 equivalents (0.65
mmol) of the mixed anhydride 50, and the reaction stirred at room temperature. After 1 hour, the
reaction was considered complete according to LC-MS analysis. The solvent was removed under
vacuum and the raw product is purified through RP-HPLC.
Characterization Yield : 27.3 mg (22.6%) as yellow powder Analytical Data : C28H31N3O9 (MW = 553,57) APCI-MS m/z = 555.2 [M+1]+ HPLC : tr = 15.8 min. purity > 99 % (254nm) IR (film) : 3530-3100, 2973, 2943, 2878, 2103, 1672, 1616, 1525, 1428, 1242, 1200,
1134, 1042 cm-1
Experimental Part
152
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.91 (m, 2H, central CH2 linker), 2.26-2.33 (m, 3H, H-≡,
CH2CONH), 2.57 (dd, 1H, J = 12.0, 8.3 Hz, H-5a), 2.59 (t, 2H, J = 7.6 Hz, ≡-CH2CH2), 2.75 (m, 1H, H-
6), 2.82 (d, 1H, J = 11.3, H-4a), 2.95 (s, 6H, N(CH3)2), 3.56 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 4.41 (s,
1H, H-4), 6.93 (d, 1H, J = 8.3 Hz, H-7), 8.15 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (150 MHz, CD3OD) :
δ (ppm) = 16.12 (CH3), 18.63 (CH2-≡), 25.77 (central CH2 linker), 36.39, 39.80, 43.04, 47.94 (C-4a,
CH2CO, C-6), 47.99 (N(CH3)2, C-5a), 67.13 (C-4), 70.02, 70.29 (C-5 and ≡C-H), 74.66 (C-12a), 84.18
(CH2-C≡), 96.22 (C-2), 108.52 (C-11a), 116.13, 116.88 (C-7,C-10a), 126.64, 130.16 (C-9, C-8),
144.47 (C-6a), 154.12 (C-10), 172.92, 174.08, 174.14 (C-12, CONH2, CONH), 195.63 (C1, C3, C11).
Ethyl 5-azidopentanoate (52)
0.158 mL (1mmol) of ethyl 5-bromo valerate were added via syringe to 3 mL of a 0.5M solution of
sodium azide in DMSO. The vial was sealed and irradiated with microwaves for 30 minutes at 100°C.
After cooling, water (50 ml) was added and the mixture extracted with ether (3 x I0 ml). The ether
extracts were washed with brine (30 ml) and dried over Na2SO4. The solvent was removed in vacuo
and the crude oil thus obtained was used without further purification.
Yield : 171 mg (100%) as colorless oil Analytical Data : C7H13N3O2 (MW = 171.20) IR (film) : 2942, 2877, 2098, 1723, 1454, 1415, 1276 cm-1
1H NMR (360 MHz, CDCl3) :
δ (ppm) = 1.25 (t, 3H, J = 7.1 Hz, CH3 ethyl ester), 1.58-1.76 (m, 4H, central CH2 linker), 2.34 (t, 2H, J
= 7.15 Hz, C-2), 3.31 (t, 2H, J = 6.5 Hz, CH2N3), 4.12 (q, 2H, J = 7.1 Hz, CH2 ethyl ester).
Experimental Part
153
5-Azidopentanoic acid (52a)
To 1 mmol of the azidoesters 52 were added 1.2 ml of a 1N aqueous solution of NaOH (1.2 mmol, 1.2
equivalents) and the minimum of methanol to make the reaction mixture homogenous. After 4 hours at
room temperature, the methanol was removed in vacuo. The aqueous solution was extracted with
ether (2 x 10 ml) and acidified to pH ~ 0 with concentrated HCI. The acids were then extracted with
ether (2 x 20 ml) and the organic phase dried over Na2SO4. After filtration and removal of the solvent
in vacuo, the crude azido acid was obtained.
Yield : 117 mg (82%) as light yellow oil Analytical Data : C5H9N3O2 (MW = 143.14) IR (film) : 2942, 2877, 2098, 1708, 1454, 1415, 1276 cm-1
1H NMR (360 MHz, CDCl3) :
δ (ppm) = 1.60-1.78 (m, 4H, central CH2 linker), 2.40 (t, 2H, J = 7.15 Hz, C-2), 3.31 (t, 2H, J = 6.5 Hz,
CH2N3).
9-(5-azidopentanamido)-Doxycycline (53)
To a solution of 200 mg (0.435 mmol) of amino derivative 39 in 5 mL of DMF, containing 2 equivalents
of NaHCO3 (0.73 mmol, 87 mg), 2 equivalents of 5-azidopentanoic acid symmetric anhydride (0.87
mmol) obtained from 52a with the described general procedure (p.140) and solubilized in DCM/DMF
(1+1 mL) were slowly dropped in. The reaction proceeded to completeness at room temperature
whithin 2 hours, as confirmed by LC-MS analysis. The solvent is then removed in vacuo and the raw
product is purified through RP-HPLC.
Characterization Yield : 270 mg (92%) as yellow powder Analytical Data : C27H32N6O9 (MW = 584,59)
Experimental Part
154
APCI-MS m/z = 568.7 [M+1-NH3]+ HPLC : tr = 17.1 min. purity > 99 % (254nm) IR (film) : 3500-3200 (OH), 2954 (alkyl chain), 2098 (N3), 1673, 1616,
1527 (C=O, Amide and C=C) cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.69 (tt, 2H, J = 7.4, 7.2 Hz, CH2CH2CO), 1.79 (tt, 2H,
J = 7.5, 7.5 Hz, CH2CH2N3), 2.51 (t, 2H, J = 7.6 Hz, CH2CO), 2.56 (dd, 1H, J = 12.3, 8.5 Hz, H-5a),
2.74 (dq, 1H, J = 13.2, 6.3 Hz, H-6), 2.81 (d, 1H, J = 11.3, H-4a), 2.95 (s, 6H, N(CH3)2), 3.36 (t, 2H J =
6.8 Hz, CH2-N3), 3.56 (dd, 1H, J = 11.5, 8.5 Hz, H-5), 4.41 (s, 1H, H-4), 6.93 (d, 1H, J = 8.3 Hz, H-7),
8.15 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.12 (CH3), 24.01 (CH2CH2CO), 29.41 (CH2CH2N3), 36.97, 39.79, 43.03, 43.09, 47.98 (C-
4a, CH2CO, C-6, N(CH3)2, C-5a), 52.20 (CH2-N3), 67.15 (C-4), 70.03 (C-5), 74.66 (C-12a), 96.29 (C-
2), 108.51 (C-11a), 116.14, 116.88 (C-7, C-10a), 126.61, 130.20 (C-9, C-8), 144.50 (C-6a), 154.15 (C-
10), 172.93, 174.07, 174.43 (C-12, CONH2, CONH), 188.10, 195.60 (C1, C3, C11).
9-[4-(1-benzyl-1H-1,2,3-triazol-4-yl)butanamido] doxycycline (54)
30 mg of derivative 51 (MW = 553.57, 0.054 mmol), 13 eq of CuI (0.715 mmol, 135 mg) and 7 eq of
ascorbic acid (0.38 mmol, 68 mg) were solved in 4 mL of dry DMF. To this solution were added firstly
benzyl azide (4 eq, 0.21 mmol, 29 mg) and then 17 eq of DIPEA (0.935 mmol, 0.16 mL). After a 2 min
treatment in a bath sonicator, the reaction mixture was heated at 40°C in oil bath. Following LC-MS
analysis, the reaction was complete within 1 hour. The mixture was diluted with 10 mL methanol,
filtered and solvents evaporated in vacuo. The crude product was dissolved in MeOH/HCl and purified
through RP-HPLC.
Characterization Yield : 18.4 mg (49%) as yellow powder Analytical Data : C35H38N6O9 (MW = 686,73)
Experimental Part
155
APCI-MS m/z = 687.3 [M+1]+ HPLC : tr = 16.8 min. purity > 99 % (254nm) IR (film) : 3500-3180 (OH), 3075, 2977, 2952, 2875, 1674, 1614, 1525 (C=O, Amide
and C=C), 1428, 1242, 1201, 1132, 1040 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.53 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.05 (tt, 2H, J = 7.4, 7.4 Hz, central CH2 side chain),
2.50 (t, 2H, J = 7.4 Hz, CH2CO), 2.55 (dd, 1H, J = 12.5, 8.3 Hz, H-5a), 2.73 (dq, 1H, J = 13.8, 6.6 Hz,
H-6), 2.79 (t, 2H J = 7.7 Hz, CH2-CN), 2.81 (d, 1H, J = 11.3, H-4a), 2.95 (s, 6H, N(CH3)2), 3.56 (dd,
1H, J = 11.3, 8.3 Hz, H-5), 4.42 (s, 1H, H-4), 5.54 (s, 2H, CH2 benzyl), 6.91 (d, 1H, J = 8.3 Hz, H-7),
7.28-7.38 (m, 5H, Phe), 7.77 (s, 1H, H-= triazole), 8.13 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.13 (CH3), 25.69 (CH2-CN), 26.48 (central CH2 side chain), 36.83 (CH2CO), 39.78 (C-6),
43.03 (C-4a), 47.96 (N(CH3)2, C-5a), 54.20 (CH2-N-N), 67.09 (C-4), 70.00 (C-5), 74.63 (C-12a), 96.15
(C-2), 108.51 (C-11a), 116.13, 116.85 (C-7, C-10a), 123.53 (H-C= triazole), 126.63, 129.07, 129.54,
130.01, 130.16, 136.8 (C-9, C-8, Aromatic Cs), 144.44 (C-6a), 148.82 (N-C=CH), 154.10 (C-10),
172.90, 174.09, 174.24 (C-12, CONH2, CONH), 188.10, 195.60 (C1, C3, C11).
52 HSQC shows the 13C chemical shifts for position 5 of the triazole and for the methylene of the benzyl rest.
Experimental Part
156
52 HMBC shows the correlation between position 5 of the triazole and the methylenic CH2 of the benzyl rest.
Ethyl 5- [4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl] pentanoate (55)
10 mg of derivative 51 (MW = 553.57, 0.018 mmol), 13 eq of CuI (0.235 mmol, 44 mg) and 7 eq of
ascorbic acid (0.13 mmol, 22 mg) were solved in 4 mL of dry DMF. To this solution were added 2
equivalents (0.036 mmol, 6 mg) of azido derivative 52 and then 17 eq of DIPEA (0.306 mmol, 0.05
mL). After a 2 min treatment in a bath sonicator, the reaction mixture was heated at 40°C in oil bath.
Following LC-MS analysis, the reaction was complete within 1 hour. The mixture was diluted with 10
mL methanol, filtered and the solvents evaporated in vacuo. The crude product was dissolved in
MeOH/HCl and purified through RP-HPLC.
Characterization Yield : 8 mg (61%) as yellow film
Experimental Part
157
Analytical Data : C35H44N6O11 (MW = 724,77) APCI-MS m/z = 725.5 [M+1]+ HPLC : tr = 16.8 min. purity > 99 % (254nm) IR (film) : 3550-3160 (OH), 3076, 2953, 2878, 1726, 1679, 1612, 1525 (C=O, Amide
and C=C), 1428, 1242, 1200, 1132, 1036 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.22 (t, 0.6H, J = 7.2 Hz, CH3 Ethyl esther), 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.59 (tt,
2H, J = 7.6, 7.6 Hz, CH2CH2N-N), 1.92 (tt, 2H, J = 7.5, 7.6 Hz, CH2CH2COOEt), 2.08 (tt, 2H, J = 7.4,
7.4 Hz, CH2CH2CONH), 2.36 (t, 2H, J = 7.4 Hz, CH2-COOEt), 2.52 (t, 2H, J = 7.4 Hz, CH2CO), 2.57
(dd, 1H, J = 12.5, 8.3 Hz, H-5a), 2.75 (dq, 1H, J = 12.8, 6.4 Hz, H-6), 2.81 (t, 2H J = 7.6 Hz, CH2-C-N),
2.82 (d, 1H, J = 11.7, H-4a), 2.96 (s, 6H, N(CH3)2), 3.56 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 3.63 (s, 2.2H,
CH3 methyl esther), 4.10 (q, 0.9H, J = 7.2 Hz, CH2 ethyl esther), 4.38 (t, 2H, J = 7.4 Hz, CH2N-N), 4.42
(s, 1H, H-4), 6.94 (d, 1H, J = 8.3 Hz, H-7), 7.81 (s, 1H, H-= triazole), 8.15 (d, 1H, J = 8.3 Hz, H-8).
(After preparative HPLC the compound reveal to be a mixture of methyl and ethyl esther, probably
dued to acidic MeOH used in the mobile phase).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 14.50 (CH3 Ethyl esther), 16.13 (CH3), 22.83 (CH2CH2COOEt), 25.66, 26.50 (CH2CONH,
CH2CH2N), 30.55 (CH2C-N), 33.91, 34.22, 36.82 (CH2COOEt, CH2CONH, C-4a), 39.80 (C-6), 43.07
(N(CH3)2), 48.02 (C-5a), 52.06 (CH2-N-N), 61.52 (CH3CH2O), 67.15 (C-4), 70.02 (C-5), 74.66 (C-12a),
96.22 (C-2), 108.52 (C-11a), 116.15, 116.90 (C-7, C-10a), 123.48 (H-C= triazole), 126.66, 130.22 (C-
9, C-8), 144.49 (C-6a), 148.40 (N-C=CH), 154.14 (C-10), 172.91, 174.10, 174.26, 175.34 (C-12,
CONH2, CONH, COOEt), 188.09, 195.62 (C1, C3, C11).
Methyl 2- [4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl]-3-phenylpropanoate (56)
20 mg of derivative 51 (MW = 553.57, 0.036 mmol), 13 eq of CuI (0.47 mmol, 89 mg) and 7 eq of
ascorbic acid (0.25 mmol, 44 mg) were solved in 4 mL of dry DMF. To this solution were added 2
equivalents of methyl 2-azido-3-phenylpropanoate (phenylalanine azide methyl ester, gently gift of Dr.
Paul), and 17 eq of DIPEA (0.614 mmol, 0.1 mL). After a 2 min treatment in a bath sonicator, the
Experimental Part
158
reaction mixture was heated at 40°C in oil bath. Following LC-MS analysis, the reaction was complete
within 1 hour. The mixture was diluted with 10 mL methanol, filtered and solvents evaporated in vacuo.
The crude product was dissolved in MeOH/HCl and purified through RP-HPLC.
Characterization Yield : 10 mg (37%) as yellow film Analytical Data : C38H42N6O11 (MW = 758,79) APCI-MS m/z = 759.5 [M+1]+ HPLC : tr = 17.4 min. purity > 95 % (254nm) IR (film) : 3500-3200 (OH), 2919, 2881 (alkyl chain), 1670, 1612, 1527 (C=O, Amide
and C=C), 1427, 1241, 1199, 1037 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.54 (d, 3H, J = 6.4 Hz, CH3 at C-6), 1.95-2.05 (m, 2H, central CH2 side chain), 2.39-2.49
(m, 2H, CH2CO), 2.53-2.60 (m, 1H, H-5a), 2.70-2.79 (m, 3H, H-6 and CH2C-N), 2.82 (d, 1H, J = 10.6,
H-4a), 2.95 (s, 6H, N(CH3)2), 3.46 and 3.53-3.63 (dd and m, 1 and 2H, diasterotopic CH2 and H-5),
3.77 (s, 3H CH3 methyl esther), 4.42 (s, 1H, H-4), 5.65-5.60 (m, 1H, C alpha azido acid), 6.94 (d, 1H, J
= 8.3 Hz, H-7), 7.05-7-25 (m, 5H, Phe), 7.81 (s, 1H, H-= triazole), 8.56 (d, 1H, J = 8.3 Hz, H-8).
57
10 mg of derivative 51 (MW = 553.57, 0.018 mmol), 13 eq of CuI (0.235 mmol, 44 mg) and 7 eq of
ascorbic acid (0.13 mmol, 22 mg) were solved in 4 mL of dry DMF. To this solution were added 2
equivalents (0.036 mmol, 8.4 mg) of azido mini-peg and then 17 eq of DIPEA (0.306 mmol, 0.05 mL).
After a 2 min treatment in a bath sonicator, the reaction mixture was heated at 40°C in oil bath.
Following LC-MS analysis, the reaction was complete within 1 hour. The mixture was diluted with 10
mL methanol, filtered and the solvents evaporated in vacuo. The crude product was dissolved in
MeOH/HCl and purified through RP-HPLC.
Characterization Yield : 4 mg (28%) as dark brown oil
Experimental Part
159
Analytical Data : C37H48N6O14 (MW = 800,83) APCI-MS m/z = 801.4 [M+1]+ HPLC : tr = 15.6 min. purity 89 % (254nm) IR (film) : 3660-3000 (OH), 2931, 2881 (alkyl chain), 2595, 1731, 1668, 1634 (C=O, Amide
and C=C), 1438, 1244, 1194, 1115 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.56(d, 3H, J = 6.8 Hz, CH3 at C-6), 2.09 (br s, 2 H, CH2CH2CONH), 2.37 (t, 2H, J = 7.4 Hz,
CH2-COOCH3), 2.54 (br s, 2H, CH2CO), 2.60 (dd, 1H, J = 12.1, 8.7 Hz, H-5a), 2.74-2.80 (m, 1H, H-6),
2.80-2.87 (m, 3H, H-4 and CH2-C-N), 2.97 (br s, 6H, N(CH3)2), 3.59-3.63 and 3.64-3-68 (2 m, 11 H,
CH2CH2-N-N, 2 x OCH2CH2O and H-5), 3.72 (s, 3H, CH3 methyl esther), 3.90 (t, 2H, J = 5.3 Hz, CH2-
N-N), 4.15 (s, 2H, OCH2COOCH3), 4.43 (s, 1H, H-4), 4.56 (br s, 2H, CH2COOCH3), 6.97 (d, 1H, J =
8.3 Hz, H-7), 7.91 (s, 1H, H-= triazole), 8.18 (d, 1H, J = 8.3 Hz, H-8).
(weak peaks and unsolved due to low concentration)
4-[1-[5-(doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-triazol-4-yl] butanoic acid
(58)
20 mg of derivative 53 (MW = 584.59, 0.034 mmol), 13 eq of CuI (0.44 mmol, 84 mg) and 7 eq of
ascorbic acid (0.24 mmol, 42 mg) were solved in 4 mL of dry DMF. To this solution were added 2
equivalents (0.068 mmol, 0.007 mL) of 5-hexynoic acid and 17 eq of DIPEA (0.578 mmol, 0.1 mL).
After a 2 min treatment in a bath sonicator, the reaction mixture was heated at 40°C in oil bath.
Following LC-MS analysis, the reaction was complete within 1 hour. The mixture was diluted with 10
mL methanol, filtered and the solvents evaporated in vacuo. The crude product was dissolved in
MeOH/HCl and purified through RP-HPLC.
Characterization Yield : 15 mg (65%) as brown glas Analytical Data : C34H42N6O11 (MW = 710,74) APCI-MS m/z = 711.3 [M+1]+ HPLC : tr = 16.0 min. purity > 95 % (254nm)
Experimental Part
160
IR (film) : 3500-3200 (OH), 2938, 2869 (alkyl chain), 1716, 1671, 1612 (C=O, Amide
and C=C), 1436, 1294, 1242, 1186, 1054 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.55 (d, 3H, J = 6.6 Hz, CH3 at C-6), 1.71 (tt, 2H, J = 7.6, 7.6 Hz, CH2CH2CONH), 1.93-2.04
(m, 4H, CH2CH2COOCH3 and CH2CH2N-N), 2.37 (t, 2H, J = 7.4 Hz, CH2-COOCH3), 2.51 (t, 2H, J =
7.4 Hz, CH2CO), 2.58 (dd, 1H, J = 12.1, 8.3 Hz, H-5a), 2.73 (t, 2H J = 7.6 Hz, CH2-C-N), 2.76 (dq, 1H,
J = 13.5, 6.4 Hz, H-6), 2.82 (d, 1H, J = 11.7, H-4a), 2.96 (br s, 6H, N(CH3)2), 3.56 (dd, 1H, J = 11.3,
8.3 Hz, H-5), 3.64 (s, 3H, CH3 methyl esther), 4.41 (s, 1H, H-4), 4.43 (t, 2H, J = 7.0 Hz, CH2N-N), 6.95
(d, 1H, J = 8.3 Hz, H-7), 7.78 (s, 1H, H-= triazole), 8.13 (d, 1H, J = 8.3 Hz, H-8).
Tert-butyl [1-[5-(doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-triazol-4-yl] methylcarbamate (59)
20 mg of derivative 53 (MW = 584.59, 0.034 mmol), 13 eq of CuI (0.44 mmol, 84 mg) and 7 eq of
ascorbic acid (0.24 mmol, 42 mg) were solved in 4 mL of dry DMF. To this solution were added 2
equivalents (0.068 mmol, 10 mg) of N-Boc-propargylamine and 17 eq of DIPEA (0.578 mmol, 0.1 mL).
After a 2 min treatment in a bath sonicator, the reaction mixture was heated at 40°C in oil bath.
Following LC-MS analysis, the reaction was complete within 1 hour. The mixture was diluted with 10
mL methanol, filtered and the solvents evaporated in vacuo. The crude product was dissolved in
MeOH/HCl and purified through RP-HPLC.
Characterization Yield : 14 mg (59%) as dark yellow film Analytical Data : C35H45N7O11 (MW = 739,79) APCI-MS m/z = 740.4 [M+1]+ HPLC : tr = 16.8 min. purity > 95 % (254nm) IR (film) : 3700-3100 (OH), 2975, 2942, 2875 (alkyl chain), 1693, 1681, 1612 (C=O, Amide
and C=C), 1529, 1428, 1244, 1200, 1177, 1133, 1057 cm-1
1H NMR (600 MHz, CD3OD) :
Experimental Part
161
δ (ppm) = 1.43 (s, 9H, tBu Boc), 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.67-1.75 and 1.96-2.04 (2 m,
each 2H, central CH2 side chain), 2.50 (t, 2H, J = 7.2 Hz, CH2CO), 2.57 (dd, 1H, J = 12.3, 8.5 Hz, H-
5a), 2.76 (dq, 1H, J = 13.1, 6.4 Hz, H-6), 2.82 (d, 1H, J = 11.3, H-4a), 2.95 (s, 6H, N(CH3)2), 3.56 (dd,
1H, J = 11.3, 8.3 Hz, H-5), 4.29 (s, 2H, NH-CH2-=), 4.41 (s, 1H, H-4), 4.44 (t, 2H, J = 6.8 Hz,CH2-N-N),
6.94 (d, 1H, J = 8.3 Hz, H-7), 7.84 (s, 1H, H-= triazole), 8.14 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.13 (CH3), 23.63 (CH2-CH2-CONH), 28.73 (CH3 Boc), 30.71 (CH2CH2N-N), 36.70
(CH2CONH), 39.81 (C-6), 43.06 (C-4a), 48.04 (N(CH3)2, C-5a, CH2-=), 51.00 (CH2-N-N), 67.11 (C-4),
69.98 (C-5), 74.63 (C-12a), 80.40 (C(CH3)3), 97.13 (C-2), 108.51 (C-11a), 116.14, 116.92 (C-7, C-
10a), 123.89 (H-C= triazole), 126.60, 130.32 (C-9, C-8), 140.85 (N-C=CH), 144.57 (C-6a), 154.13 (C-
10), 158.18 (NHCOOtBu), 172.14, 174.01, 174.14 (C-12, CONH2, CONH), 187.80, 195.56 (C1, C3,
C11).
9-[5-[4-(Aminomethyl)-1H-1,2,3-triazol-1-yl] pentanamido] doxycycline (60)
10 mg of derivative 53 (MW = 584.59, 0.034 mmol), 13 eq of CuI (0.44 mmol, 84 mg) and 7 eq of
ascorbic acid (0.24 mmol, 42 mg) were solved in 4 mL of dry DMF. To this solution were added 2
equivalents (0.068 mmol, 0.004 mL) of propargylamine and 17 eq of DIPEA (0.578 mmol, 0.1 mL).
After a 2 min treatment in a bath sonicator, the reaction mixture was heated at 40°C in oil bath.
Following LC-MS analysis, the reaction was complete within 1 hour. The mixture was diluted with 10
mL methanol, filtered and the solvents evaporated in vacuo. The crude product was dissolved in
MeOH/HCl and purified through RP-HPLC.
Characterization Yield : 7.6 mg (35%) as yellow film Analytical Data : C30H37N7O9 (MW = 639,67) APCI-MS m/z = 640.4 [M+1]+ HPLC : tr = 11.9 min. purity > 95 % (254nm) 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.55 (d, 3H, J = 6.4 Hz, CH3 at C-6), 1.66-1.74 and 1.98-2.05 (2 m, each 2H, central CH2
side chain), 2.52 (t, 2H, J = 7.2 Hz, CH2CO), 2.58 (dd, 1H, J = 12.3, 8.1 Hz, H-5a), 2.76 (m, 1H, H-6),
Experimental Part
162
2.82 (d, 1H, J = 11.7, H-4a), 2.95 (s, 6H, N(CH3)2), 3.57 (dd, 1H, J = 11.4, 8.5 Hz, H-5), 4.24 (s, 2H,
NH-CH2-=), 4.41 (s, 1H, H-4), 4.50 (t, 2H, J = 7.0 Hz,CH2-N-N), 6.95 (d, 1H, J = 8.3 Hz, H-7), 8.07 (s,
1H, H-= triazole), 8.13 (d, 1H, J = 8.3 Hz, H-8).
General procedure for the synthesis of N-terminus modified amino acid building blocks : 1.25 g of 2-chlorotrityl resin (loading 1.6 mmol/g) were swollen in 10 mL DCM. 0.26 mmol of Fmoc-
protected amino acid and 4 equivalents of DIPEA solubilized in DCM, were added and the attachment
proceeded at room temperature for 2h. The resin was separated from the solution by filtration and
washed 3 times with a mixture of DCM/MeOH/DIPEA (17:2:1), then 5 times with DMF and finally §
times with acid-free DCM. The Fmoc-amino acid loaded resin was divided into 2 parts, and each
coupled with the respective azido or alkyne linker.
Fmoc deprotection was afforde treating the resin with 20% piperidine in DMF (microwave irradiation:
5x5s, 100W), followed by washings with DMF (5x). Peptide coupling was done employing 5 eq. of the
corresponding linker / PyBOP / DIPEA and 7.5 eq. HOBt, dissolved in a minimum amount of DMF
(irradiation: 15x10s, 50W). In between each irradiation step, cooling of the reaction mixture to a
temperature of -10°C was achieved by sufficient agitation in an ethanol-ice bath.
The cleavage from the resin was performed using a mixture of TFA/phenol/H2O/triisopropylsilane
88:5:5:2 for 2h, followed by a filtration of the resin. After evaporation of the solvent in vacuo and
precipitation in t-butylmethylether, the crude peptides were used for the click reaction without further
purifications.
2-hex-5-ynamidoacetic acid (61)
Compound 61 was synthesized according to the general procedure starting from 137.5 mg resin (0.22
mmol).
Characterization Yield : 32 mg (86%) as colorless oil Analytical Data : C8H11NO3 (MW = 169,18) IR (film) : 3289, 2935, 1963, 1731, 1666, 1542, 1388, 1099 cm-1
(S)-2-hex-5-ynamidopropanoic acid (62)
Experimental Part
163
Compound 62 was synthesized according to the general procedure starting from 100 mg resin (0.16
mmol).
Characterization Yield : 26 mg (89%) as colorless oil Analytical Data : C9H13NO3 (MW = 183,20) IR (film) : 3293, 2938, 2117, 1731, 1643, 1546, 1454, 1214, 1168, 651 cm-1 1H NMR (360 MHz, CDCl3) :
δ (ppm) = 1.47 (d, 3H, J = 7.0 Hz, CH3 Ala), 1.87 (tt, 2H, J = 7.1, 7.2 Hz, CH2CH2CONHAla), 2.02 (t,
2H, J = 2.6 Hz, CH alkyne), 2.28 (dt, 2H, J = 6.9, 2.6 Hz, CH2-4), 2.41 (t, 2H, J = 7.4 Hz, CH2-
CONHAla), 4.60 (dq, 1H, J = 7.2, 7.3 Hz, H alpha Ala), 6.55 (d, 1H, J = 7.3 Hz, NH), 8.60-8.80 (br s,
1H, COOH).
13C NMR (90 MHz, CDCl3) :
δ (ppm) = 17.71 (CH3 Ala), 17.97 (C-4), 24.03 (CH2CH2CONHPhe), 34.74 (CH2CONHAla), 48.22 (C
alpha), 69.38 (C-H alkyne), 83.26 (C-5 alkyne), 173.19, 175.78 (CONH2, COOH).
(S)-2-hex-5-ynamido-3-phenylpropanoic acid (63)
Compound 63 was synthesized according to the general procedure starting from 100 mg resin (0.16
mmol).
Characterization Yield : 38 mg (92%) as colorless oil Analytical Data : C15H17NO3 (MW = 259,30) IR (film) : 3293, 3031, 2935, 2117, 1731, 1646, 1542, 1438, 1384, 1219, 701, 667 cm-1 1H NMR (360 MHz, CDCl3) :
Experimental Part
164
δ (ppm) = 1.79 (dt, 2H, J = 14.9, 7.5 Hz, CH2CH2CONHPhe), 1.95 (t, 2H, J = 2.6 Hz, CH alkyne), 2.12-
2.23 (m, 2H, CH2-4), 2.32 (t, 2H, J = 7.0 Hz, CH2-CONHAla), 3.11 and 3.24 (2 dd, 1H each, J = 6.7,
14.0 Hz, CH2 Phe), 4.87 (dt, 1H, J = 6.4, 6.6 Hz, H alpha Phe), 6.15 (d, 1H, J = 7.5 Hz, NH), 7.15-7.32
(m, 5H, aromatics), 7.60-8.20 (br s, 1H, COOH).
13C NMR (90 MHz, CDCl3) :
δ (ppm) = 17.71 (C-4), 24.04 (CH2CH2CONHPhe), 34.74, 37.39 (CH2CONHPhe, CH2 Phe), 53.28 (C
alpha), 69.41 (C-H alkyne), 83.31 (C-5 alkyne), 127.20, 128.67, 129.37, 135.83 (Aromatics), 173.01,
174.57 (CONH2, COOH).
(2-(5-azidopentanamido) acetic acid (64)
Compound 64 was synthesized according to the general procedure starting from 200 mg resin (0.33
mmol).
Characterization Yield : 46.4 mg (70%) as colorless oil Analytical Data : C7H12N4O3 (MW = 200.20) IR (film) : 3313, 2938, 2873, 2098, 1735, 1646, 1546, 1207 cm-1
(S)-2-(5-azidopentanamido) propanoic acid (65)
Compound 65 was synthesized according to the general procedure starting from 200 mg resin (0.33
mmol).
Characterization Yield : 65.5 mg (92%) as colorless oil Analytical Data : C8H14NO3 (MW = 214,22) IR (film) : 3309, 2942, 2098, 1727, 1646, 1542, 1454, 1234 cm-1 1H NMR (600 MHz, CDCl3) :
Experimental Part
165
δ (ppm) = 1.44 (d, 3H, J = 7.2 Hz, CH3 Ala), 1.62 (tt, 2H, J = 7.2, 7.4 Hz, CH2CH2N3), 1.71 (tt, 2H, J =
7.6, 7.4 Hz, CH2CH2CONH), 2.29 (t, 2H, J = 7.6 Hz, CH2-CONHAla), 3.29 (t, 2H, J = 6.8 Hz, CH2-N3),
4.57 (dq, 1H, J = 7.2, 7.3 Hz, H alpha Ala), 6.57 (d, 1H, J = 7.2 Hz, NH), 9.76-10.73 (br s, 1H, COOH).
13C NMR (90 MHz, CDCl3) :
δ (ppm) = 17.98 (CH3 Ala), 22.64 (C-3), 28.12 (CH2CH2N3), 35.48 (CH2CONHAla), 48.18 (C alpha),
50.98 (CH2N3), 173.26, 175.59 (CONH2, COOH).
(S)-2-(5-azidopentanamido)-3-phenylpropanoic acid (66)
Compound 66 was synthesized according to the general procedure starting from 200 mg resin (0.33
mmol).
Characterization Yield : 74 mg (78%) as colorless oil Analytical Data : C14H18N4O3 (MW = 290,32) IR (film) : 3309, 3062, 3031, 2935, 2869, 1731, 1650, 1261, 1099 cm-1 1H NMR (360 MHz, CDCl3) :
δ (ppm) = 1.47-1.69 (m, 4H, central CH2 linker), 2.20 (dt, 2H, J = 1.8, 7.0 Hz, CH2-CONHPhe), 3.10
and 3.24 (2 dd, 1H each, J = 6.7, 14.0 Hz, CH2 Phe), 3.23 (t, 2H, J = 6.6 Hz, CH2-N3), 4.87 (dt, 1H, J =
6.4, 6.8 Hz, H alpha Phe), 6.11 (d, 1H, J = 7.5 Hz, NH), 7.12-7.32 (m, 5H, aromatics), 7.35-7.65 (br s,
1H, COOH).
13C NMR (90 MHz, CDCl3) :
δ (ppm) = 22.62, 28.08 (central CH2 linker), 35.55, 37.32 (CH2CONHPhe, CH2 Phe), 51.01 (CH2N3),
53.23 (C alpha), 127.19, 128.62, 129.27, 135.79 (Aromatics), 173.16, 174.69 (CONH2, COOH).
Experimental Part
166
General procedure for the click reaction with amino acid building blocks 61-66 : 1 equivalent of the doxycycline derivative (51 or 53), 13 equivalents of copper iodide, 7 equivalents of
ascorbic acid were dissolved in dry DMF degassed and charged with N2. To this mixture 2 equivalents
of amino acid modified building block (61-66) dissolved in the minimum amount of dry DMF were
added and finally 17 equivalents of DIPEA were added via syringe. The vial was sonicated for 30
seconds to better solubilize the catalyst, and then 4 cycles of 30” microwave irradiation (100W power,
50°C temperature limit) were operated. Between every cycle the vial was cooled to circa -10°C in an
acetone/ice bath. The mixture was diluted with H20 and lyophilized, in order to remove DMF. The
crude mixture was recovered with acidic (HCl) THF, permitting the filtration of the copper catalyst. THF
was evaporated at reduced pressure, the crude product solubilized in acetonitrile and purified through
reverse phase HPLC yielding pure compounds.
2-[5-[4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl] pentanamido]
acetic acid (67)
20 mg of derivative 51 (MW = 553.57, 0.036 mmol), 13 eq of CuI (0.47 mmol, 89 mg) and 7 eq of
ascorbic acid (0.25 mmol, 44 mg) were solved in 2 mL of dry DMF. To this solution were added 2
equivalents of 64 (MW = 200.20, 14 mg) in 1 ml of dry DMF, and 17 eq of DIPEA (0.614 mmol, 0.1
mL). Reaction and purification followed the general procedure.
Characterization Yield : 14.6 mg (54%) as yellow film Analytical Data : C35H43N7O12 (MW = 753,77) APCI-MS m/z = 754.4 [M+1]+ HPLC : tr = 14.6 min. purity > 95 % (254nm) IR (film) : 3500-3200 (OH), 2931, 2865 (alkyl chain), 1673, 1616, 1531 (C=O,
Amide and C=C), 1430, 1241, 1199, 1133 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.55 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.62 (tt, 2H, J = 7.6, 7.6 Hz, CH2CH2COGly), 1.95 (tt,
2H, J = 7.5, 7.6 Hz, CH2CH2N-N), 2.08 (tt, 2H, J = 7.4, 7.4 Hz, CH2CH2CONHDoxy), 2.29 (t, 2H, J =
7.2 Hz, CH2-COGly), 2.52 (t, 2H, J = 7.4 Hz, CH2CONHDoxy), 2.58 (dd, 1H, J = 12.1, 8.3 Hz, H-5a),
2.76 (dq, 1H, J = 13.5, 6.4 Hz, H-6), 2.82 (t, 2H J = 7.2 Hz, CH2-C-N), 2.83 (d, 1H, J = 11.8, H-4a),
Experimental Part
167
2.95 (br s, 6H, N(CH3)2), 3.56 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 3.88 (s, 2H, CH2 glycine), 4.40 (t, 2H, J =
7.0 Hz, CH2N-N), 4.42 (s, 1H, H-4), 6.95 (d, 1H, J = 8.3 Hz, H-7), 7.83 (s, 1H, H-= triazole), 8.14 (d,
1H, J = 8.3 Hz, H-8).
(S)-2-[5-[4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl] pentanamido] propanoic acid (68)
20 mg of derivative 51 (MW = 553.57, 0.036 mmol), 13 eq of CuI (0.47 mmol, 89 mg) and 7 eq of
ascorbic acid (0.25 mmol, 44 mg) were solved in 2 mL of dry DMF. To this solution were added 2
equivalents of 65 (MW = 214,22, 15 mg)) in 1 mL of dry DMF, and 17 eq of DIPEA (0.614 mmol, 0.1
mL). Reaction and purification followed the general procedure.
Characterization Yield : 5.5 mg (20%) as yellow film Analytical Data : C36H45N7O12 (MW = 767,80) APCI-MS m/z = 768.3 [M+1]+ HPLC : tr = 14.9 min. purity > 99 % (254nm) IR (film) : 3500-3200 (OH), 2950 (alkyl chain), 1781, 1670, 1623, 1531 (C=O,
Amide and C=C), 1430, 1195, 1153 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.37 (d, 3H, J = 7.5 Hz, CH3 Ala), 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.61 (tt, 2H, J = 7.9,
7.2 Hz, CH2CH2CO-Ala), 1.93 (tt, 2H, J = 7.5, 7.4 Hz, CH2CH2N-N), 2.03-2.13 (m, 2H,
CH2CH2CONHDoxy), 2.27 (t, 2H, J = 6.8 Hz, CH2-CO-Ala), 2.46-2.55 (br s, 2H, CH2CONHDoxy), 2.57
(dd, 1H, J = 12.3, 8.6 Hz, H-5a), 2.72-2.85 (m, 4H, H-6, CH2-C-N, H-4a), 2.96 (br s, 6H, N(CH3)2), 3.56
(dd, 1H, J = 10.8, 8.5 Hz, H-5), 4.30-4.43 (m, 4H, CH2N-N, H alpha alanine and H-4), 6.94 (d, 1H, J =
8.2 Hz, H-7), 7.81 (s, 1H, H-= triazole), 8.15 (d, 1H, J = 8.2 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.13 (CH3), 17.55 (CH3Ala), 22.60 (CH2CH2CO-Ala), 25.69, 26.48 (CH2CONH, CH2CH2N),
30.52 (CH2C-N), 35.74, 36.81 (CH2COOEt, CH2CONH, C-4a, C-6), 42.91 (N(CH3)2), 48.29 (C-5a),
Experimental Part
168
50.92 (CH2-N-N, C alpha Ala), 70.09 (C-4), 74.82 (C-5), 80.58 (C-12a), 96.57 (C-2), 108.55 (C-11a),
116.18, 116.96 (C-7, C-10a), 123.63 (H-C= triazole), 126.70, 130.34 (C-9, C-8), 144.59 (C-6a), 148.39
(N-C=CH), 154.24 (C-10), 174.36, 175.31, 176.10 (C-12, CONH2, COOH), 181.41 (CONHDoxy),
187.93, 195.45, 195.61 (C1, C3, C11).
(S)-2-[5-[4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl] pentanamido]-3-phenylpropanoic acid (69)
20 mg of derivative 51 (MW = 553.57, 0.036 mmol), 13 eq of CuI (0.47 mmol, 89 mg) and 7 eq of
ascorbic acid (0.25 mmol, 44 mg) were solved in 2 mL of dry DMF. To this solution were added 2
equivalents of 66 (MW = 290,32, 21 mg) in 1 mL of dry DMF, and 17 eq of DIPEA (0.614 mmol, 0.1
mL). Reaction and purification followed the general procedure.
Characterization Yield : 15.3 mg (50.3%) as yellow film Analytical Data : C42H49N7O12 (MW = 843,90) APCI-MS m/z = 844.4 [M+1]+ HPLC : tr = 16.8 min. purity > 95 % (254nm) IR (film) : 3560-3050 (OH), 2933, 2876 (alkyl chain), 1743 (COOH), 1666, 1614,
1529 (C=O, Amide and C=C), 1432, 1241, 1191, 1033 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.48 (tt, 2H, J = 7.5, 7.5 Hz, CH2CH2COPhe), 1.54 (d, 3H, J = 6.4 Hz, CH3 at C-6), 1.66-
1.79 (m, 2H, CH2CH2N-N), 2.07 (tt, 2H, J = 7.4, 7.4 Hz, CH2CH2CONHDoxy), 2.14-2.23 (m, 2H, CH2-
COPhe), 2.52 (t, 2H, J = 7.2 Hz, CH2CONHDoxy), 2.56 (dd, 1H, J = 12.5, 8.3 Hz, H-5a), 2.74 (dq, 1H,
J = 13.0, 6.4 Hz, H-6), 2.80 (t, 2H J = 7.3 Hz, CH2-C-N), 2.81 (d, 1H, J = 11.8, H-4a), 2.95 (br s, 6H,
N(CH3)2), 2.90 and 3.21 (2 dd,each 1H, J = 14.0, 9.8 Hz, J = 14.0, 4.5 Hz, diasterotopic CH2 Phe),
3.56 (dd, 1H, J = 11.2, 8.4 Hz, H-5), 4.28 (t, 2H, J = 7.0 Hz, CH2N-N), 4.41 (s, 1H, H-4), 4.68 (dd, 1H,
J = 9.8, 4.2 Hz, H alpha Phe), 6.93 (d, 1H, J = 8.3 Hz, H-7), 7.14-7.25 (m, 5H, Phenyl), 7.72 (s, 1H, H-
= triazole), 8.14 (d, 1H, J = 8.3 Hz, H-8).
Experimental Part
169
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.13 (CH3), 19.94, 23.51, 25.64 (CH2Phe, CH2CH2CO-Phe, CH2C-N), 26.48, 30.31
(CH2CH2N, C-4a), 35.75, 36.76, 38.37, (CHCOOH, CH2CONH, CH2CONHDoxy), 39.77 (C-6), 42.93,
43.00 (N(CH3)2), 48.00 (C-5a), 50.83 (CH2-N-N), 54.83 (CH alpha Phe), 67.11 (C-4), 69.99 (C-5),
74.65 (C-12a), 96.22 (C-2), 108.52 (C-11a), 116.14, 116.90 (C-7, C-10a), 123.46 (H-C= triazole),
126.61, 127.76, 129.43, 130.19, 130.26, 138.59 (C-9, C-8, Phenyl Cs, N-C=CH), 144.52 (C-6a),
154.15 (C-10), 174.05, 174.28, 174.73, 175.25 (C-12, CONH2, COOH, CONHDoxy), 188.07, 195.58,
195.62 (C1, C3, C11). 2-[4-[1-[5-(doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-triazol-4-yl] butanamido]
acetic acid (70)
20 mg of derivative 53 (MW = 584.59, 0.034 mmol), 13 eq of CuI (0.44 mmol, 84 mg) and 7 eq of
ascorbic acid (0.24 mmol, 42 mg) were solved in 2 mL of dry DMF. To this solution were added 2
equivalents of 61 ((MW = 169,18, 11.5 mg) in 1 mL of dry DMF, and 17 eq of DIPEA (0.614 mmol, 0.1
mL). Reaction and purification followed the general procedure.
Characterization Yield : 12.9 mg (52%) as yellow film Analytical Data : C35H43N7O12 (MW = 753,77) APCI-MS m/z = 754.4 [M+1]+ HPLC : tr = 14.6 min. purity > 95 % (254nm) IR (film) : 3530-3190 (OH), 2957, 2878 (alkyl chain), 1741 (COOH), 1672, 1620,
1530 (C=O, Amide and C=C), 1428, 1241, 1201, 1138 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.70 (tt, 2H, J = 7.3, 7.3 Hz, CH2CH2CONHDoxy),
1.94-2.03 (m, 4H, CH2CH2N-N and CH2CH2COGly), 2.29 (t, 2H, J = 7.4 Hz, CH2-COGly), 2.51 (t, 2H, J
= 7.2 Hz, CH2CONHDoxy), 2.56 (dd, 1H, J = 11.9, 8.5 Hz, H-5a), 2.74 (br t, 3H J = 7.4 Hz, CH2-C-N
and H-6), 2.79 (d, 1H, J = 11.0, H-4a), 2.94 (br s, 6H, N(CH3)2), 3.57 (dd, 1H, J = 11.0, 8.6 Hz, H-5),
3.87 (s, 2H, CH2 glycine), 4.38 (s, 1H, H-4), 4.43 (t, 2H, J = 6.8 Hz, CH2N-N), 6.93 (d, 1H, J = 8.3 Hz,
H-7), 7.80 (s, 1H, H-= triazole), 8.11 (d, 1H, J = 8.3 Hz, H-8).
Experimental Part
170
(S)-2-[4-[1-[5-(doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-triazol-4-yl] butanamido] propanoic acid (71)
20 mg of derivative 53 (MW = 584.59, 0.034 mmol), 13 eq of CuI (0.44 mmol, 84 mg) and 7 eq of
ascorbic acid (0.24 mmol, 42 mg) were solved in 2 mL of dry DMF. To this solution were added 2
equivalents of 62 (MW = 183,20, 12.5 mg) in 1 mL of dry DMF, and 17 eq of DIPEA (0.614 mmol, 0.1
mL). Reaction and purification followed the general procedure.
Characterization Yield : 6.4 mg (24.2%) as yellow film Analytical Data : C36H45N7O12 (MW = 767,80) APCI-MS m/z = 768.3 [M+1]+ HPLC : tr = 13.2 and 13.8 min. purity > 99 % (254nm) (Double peak due to COOH-COO – equilibrium) IR (film) : 3500-3200 (OH), 2942 (alkyl chain), 1781 (COOH), 1674, 1619, 1531 (C=O,
Amide and C=C), 1427, 1195, 1137, 1037 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.37 (d, 3H, J = 7.2 Hz, CH3 Ala), 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.70 (tt, 2H, J = 7.4,
7.5 Hz, CH2CH2CO-Ala), 1.93-2.03 (m, 4H, CH2CH2N-N, CH2CH2CONHDoxy), 2.27 (t, 2H, J = 7.4 Hz,
CH2-CO-Ala), 2.51 (t, 2H, J = 7.2 Hz, CH2CONHDoxy), 2.57 (dd, 1H, J = 12.5, 8.3 Hz, H-5a), 2.71-
2.78 (m, 3H, H-6, CH2-C-N), 2.82 (d, 1H, J = 11.3 Hz, H-4a), 2.95 (br s, 6H, N(CH3)2), 3.56 (dd, 1H, J
= 11.5, 8.5 Hz, H-5), 4.36 (q, 1H, J = 7.3 Hz, H alpha Alanine), 4.41 (s, 1H, H-4), 4.43 (t, 2H, CH2N-N),
6.94 (d, 1H, J = 8.3 Hz, H-7), 7.80 (s, 1H, H-= triazole), 8.13 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.11 (CH3), 17.58 (CH3Ala), 23.65 (CH2CH2CO-Ala), 25.56, 26.52 (CH2CONH, CH2CH2N),
30.66 (CH2C-N), 35.81, 36.69 (CH2COOEt, CH2CONH, C-4a), 43.00, 43.05 (N(CH3)2, C-6), 48.01 (C-
5a), 50.93 (CH2-N-N, C alpha Ala), 67.13 (C-4), 69.99 (C-5), 74.65 (C-12a), 96.26 (C-2), 108.52 (C-
11a), 116.15, 116.91 (C-7, C-10a), 123.49 (H-C= triazole), 126.58, 130.35 (C-9, C-8, N-C=CH),
144.59 (C-6a), 154.22 (C-10), 172.93, 174.22, 175.32, 176.06 (C-12, CONH2, COOH, CONHDoxy),
188.04, 195.34, 195.62 (C1, C3, C11).
Experimental Part
171
(S)-2-[4-[1-[5-(doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-triazol-4-yl] butanamido]-3-phenylpropanoic acid (72)
20 mg of derivative 53 (MW = 584.59, 0.034 mmol), 13 eq of CuI (0.44 mmol, 84 mg) and 7 eq of
ascorbic acid (0.24 mmol, 42 mg) were solved in 2 mL of dry DMF. To this solution were added 2
equivalents of 63 (MW = 259,30, 17.6 mg) in 1 mL of dry DMF, and 17 eq of DIPEA (0.614 mmol, 0.1
mL). Reaction and purification followed the general procedure.
Characterization Yield : 15.4 mg (53.3%) as yellow film Analytical Data : C42H49N7O12 (MW = 843,90) APCI-MS m/z = 844.4 [M+1]+ HPLC : tr = 16.8 min. purity > 99 % (254nm) IR (film) : 3550-3170 (OH), 2945 (alkyl chain), 1737 (COOH), 1665,
1615, 1529 (C=O, Amide and C=C), 1431, 1241, 1033 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.52 (d, 3H, J = 6.4 Hz, CH3 at C-6), 1.69 (tt, 2H, J = 7.3, 7.3 Hz, CH2CH2CONHDoxy), 1.85
(tt, 2H, J = 7.4, 7.5 Hz, CH2CH2CONHPhe), 2.00 (tt, 2H, J = 6.8, 6.9 Hz, CH2CH2N-N), 2.17-2.22 (m,
2H, CH2-CONHPhe), 2.51 (t, 2H, J = 7.5 Hz, CH2CONHDoxy), 2.54 (dd, 1H, J = 12.5, 8.6 Hz, H-5a),
2.59 (t, 2H J = 6.8 Hz, CH2-C-N), 2.72 (dq, 1H, J = 12.0, 6.4 Hz, H-6), 2.81 (d, 1H, J = 11.3, H-4a),
2.95 (br s, 6H, N(CH3)2), 2.90 and 3.21 (2 dd,each 1H, J = 14.2, 9.6 Hz, J = 14.2, 4.9 Hz, diasterotopic
CH2 Phe), 3.56 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 3.72 (t, 2H, J = 6.6 Hz, CH2N-N), 4.41 (s, 1H, H-4),
4.68(dd, 1H, J = 9.4, 4.9 Hz, H alpha Phe), 6.91 (d, 1H, J = 8.3 Hz, H-7), 7.14-7.25 (m, 5H, Phenyl),
7.71 (s, 1H, H-= triazole), 8.12 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.11 (CH3), 23.70, 25.40,26.48 (CH2Phe, CH2CH2CO-Phe, CH2C-N), 30.65 (CH2CH2N, C-
4a), 35.83, 36.72, 38.42, (CHCOOH, CH2CONH, CH2CONHDoxy), 39.81 (C-6), 43.10 (N(CH3)2),
48.03 (C-5a), 50.83 (CH2-N-N), 54.85 (CH alpha Phe), 67.11 (C-4), 69.96 (C-5), 74.63 (C-12a), 96.18
(C-2), 108.44 (C-11a), 116.15, 116.91 (C-7, C-10a), 123.42 (H-C= triazole), 126.51, 127.74, 129.43,
Experimental Part
172
130.21, 130.37, 138.52 (C-9, C-8, Phenyl Cs, N-C=CH), 144.57 (C-6a), 154.21 (C-10), 172.87,
174.20, 174.75, 175.34 (C-12, CONH2, COOH, CONHDoxy), 188.08, 195.58 (C1, C3, C11). General method for the preparation of methyl esters from compounds 67-72 : 5-10 milligrams of free carboxylic acid clicked derivatide (67-70) were dissolved in 1 mL of
methanol.To this solution 0.1 mL of concentrated chloridric acid were added and the reaction stirred at
room temperature overnight. To this solution were then added 2 mL of H20 and directly purificated by
reverse phase HPLC, yielding the pure compounds.
Methyl 2-[5-[4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl] pentanamido] acetate (73)
The reaction followed the general procedure, starting from 10 milligrams (0.013 mmol) of derivative
67.
Characterization Yield : 8.5 mg (83%) as yellow film Analytical Data : C36H45N7O12 (MW = 767,80) APCI-MS m/z = 768.2 [M+1]+ HPLC : tr = 16.8 min. purity > 98 % (254nm) IR (film) : 3500-3120 (OH), 3066 (Aromatic H), 2952, 2861 (alkyl chain), 1734, 1667, 1634,
1597, 1550 (C=O, Amide and C=C), 1437, 1244, 1195, 1043 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.55 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.62 (tt, 2H, J = 7.6, 7.6 Hz, CH2CH2COGly), 1.95 (tt,
2H, J = 7.5, 7.6 Hz, CH2CH2N-N), 2.08 (tt, 2H, J = 7.4, 7.4 Hz, CH2CH2CONHDoxy), 2.29 (t, 2H, J =
7.2 Hz, CH2-COGly), 2.52 (t, 2H, J = 7.4 Hz, CH2CONHDoxy), 2.58 (dd, 1H, J = 12.1, 8.3 Hz, H-5a),
2.76 (dq, 1H, J = 13.5, 6.4 Hz, H-6), 2.82 (t, 2H J = 7.2 Hz, CH2-C-N), 2.83 (d, 1H, J = 11.8, H-4a),
2.93 and 2.99 (2 br s, each 3H, N(CH3)2), 3.56 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 3.69 (s, 3H, CH3 methyl
esther), 3.92 (s, 2H, CH2 glycine), 4.40 (t, 2H, J = 7.0 Hz, CH2N-N), 4.42 (s, 1H, H-4), 6.95 (d, 1H, J =
Experimental Part
173
8.3 Hz, H-7), 7.83 (s, 1H, H-= triazole), 8.14 (d, 1H, J = 8.3 Hz, H-8). Interpretation is partly based on
the following 2D spectra.
73. H-H COSY
(S)-Methyl 2-[5-[4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl] pentanamido] propanoate (74)
The reaction followed the general procedure, starting from 10 milligrams (0.013 mmol) of derivative
68.
Characterization Yield : 10.2 mg (100%) Analytical Data : C36H45N7O12 (MW = 781,83) APCI-MS m/z = 782.3 [M+1]+ HPLC : tr = 15.2 min. purity > 96 % (254nm)
Experimental Part
174
IR (film) : 3500-3140 (OH), 3020 (Aromatic), 2930, 2876 (alkyl chain), 1681, 1621, 1539
(C=O, Amide and C=C), 1432, 1201, 1136, 1037 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.36 (d, 3H, J = 7.5 Hz, CH3 Ala), 1.55 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.58-1.65 (m, 2H,
CH2CH2CO-Ala), 1.96 (tt, 2H, J = 7.4, 7.4 Hz, CH2CH2N-N), 2.08-2.14 (m, 2H, CH2CH2CONHDoxy),
2.25-2.32 (m, 2H, CH2-CO-Ala), 2.55 (t, 2H, J = 7.3 Hz, CH2CONHDoxy), 2.59 (dd, 1H, J = 12.4, 8.3
Hz, H-5a), 2.76 (dq, 1H, J = 13.2, 6.3 Hz, C-6), 2.83 (d, 1H, J = 11.3 Hz, H-4a), 2.85-2.89 (m, 2H,
CH2-C-N), 2.96 (br s, 6H, N(CH3)2), 3.57 (dd, 1H, J = 11.7, 8.3 Hz, H-5), 3.69 (s, 3H, CH3 methyl
esther), 4.37 (q, 1H, J = 7.3 Hz, H alpha alanine), 4.43 (s, 1H, H-4), 4.45 (t, 2H, J = 7.4 Hz, CH2N-N),
6.95 (d, 1H, J = 8.3 Hz, H-7), 7.04 (s, 1H, H-= triazole), 8.14 (d, 1H, J = 8.3 Hz, H-8).
(S)-Methyl 2-[5-[4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl] pentanamido]-3-phenylpropanoate (75)
The reaction followed the general procedure, starting from 10 milligrams (0.012 mmol) of derivative
69.
Characterization Yield : 9.2 mg (90.8%) Analytical Data : C43H51N7O12 (MW = 857,93) APCI-MS m/z = 858.4 [M+1]+ HPLC : tr = 16.8 min. purity > 99 % (254nm) IR (film) : 3500-3200 (OH), 3061 (Aromatic H), 2927, 2873 (alkyl chain), 1739, 1662, 1616,
1527 (C=O, Amide and C=C), 1430, 1241, 1195, 1033 cm-1 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.48 (tt, 2H, J = 7.4, 7.4 Hz, CH2CH2COPhe), 1.54 (d, 3H, J = 6.6 Hz, CH3 at C-6), 1.66-
1.79 (m, 2H, CH2CH2N-N), 2.08 (tt, 2H, J = 7.2, 7.2 Hz, CH2CH2CONHDoxy), 2.14-2.23 (m, 2H, CH2-
COPhe), 2.52 (t, 2H, J = 7.2 Hz, CH2CONHDoxy), 2.57 (dd, 1H, J = 12.3, 8.5 Hz, H-5a), 2.74 (dq, 1H,
J = 13.2, 6.3 Hz, H-6), 2.79-2.84 (m, 3H, CH2-C-N, H-4a), 2.95 (br s, 6H, N(CH3)2), 2.90 and 3.21 (2
Experimental Part
175
dd,each 1H, J = 14.0, 9.8 Hz, J = 14.0, 5.2 Hz, diasterotopic CH2 Phe), 3.56 (dd, 1H, J = 11.3, 8.3 Hz,
H-5), 3.67 (s, 3H, CH3 methyl esther), 4.30 (t, 2H, J = 7.0 Hz, CH2N-N), 4.42 (s, 1H, H-4), 4.67 (dd,
1H, J = 9.7, 5.2 Hz, H alpha Phe), 6.94 (d, 1H, J = 8.3 Hz, H-7), 7.14-7.25 (m, 5H, Phenyl), 7.75 (s,
1H, H-= triazole), 8.15 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.14 (CH3), 23.53, 25.64 (CH2Phe, CH2CH2CO-Phe, CH2C-N), 26.50, 30.35, 31.66
(CH2CH2N, C-4a), 35.70, 36.78, 38.36, (CHCOOH, CH2CONH, CH2CONHDoxy), 39.80 (C-6), 43.03
(N(CH3)2), 47.99 (C-5a), 50.89 (CH2-N-N), 52.73 (OCH3), 55.06 (CH alpha Phe), 67.12 (C-4), 70.01
(C-5), 74.64 (C-12a), 101.40 (C-2), 108.53 (C-11a), 116.15, 116.90 (C-7, C-10a), 123.51 (H-C=
triazole), 126.64, 127.87, 129.49, 130.18, 130.28, 138.28 (C-9, C-8, Phenyl Cs, N-C=CH), 144.52 (C-
6a), 154.18 (C-10), 173.61, 174.08, 174.28, 175.33 (C-12, CONH2, COOMe, CONHDoxy), 195.55,
195.62 (C1, C3, C11). Interpretation is partly based on the following 2D spectra.
75 H-H COSY
Experimental Part
176
Methyl-2-[4-[1-[5-(doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-triazol-4-yl] butanamido] acetate (76)
The reaction followed the general procedure, starting from 10 milligrams (0.013 mmol) of derivative
70.
Characterization Yield : 5.4 mg (53%) as yellow film Analytical Data : C36H45N7O12 (MW = 767,80) APCI-MS m/z = 768.2 [M+1]+ HPLC : tr = 16.8 min. purity > 97 % (254nm) IR (film) : 3500-3200 (OH), 3081 (Aromatic H), 2954, 2873 (alkyl chain), 1751, 1670, 1616,
1527 (C=O, Amide and C=C), 1430, 1241, 1195, 1141. 1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.54 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.70 (tt, 2H, J = 7.4, 7.5 Hz, CH2CH2CONHDoxy),
1.94-2.03 (m, 4H, CH2CH2N-N and CH2CH2COGly), 2.29 (t, 2H, J = 7.4 Hz, CH2-COGly), 2.51 (t, 2H, J
= 7.2 Hz, CH2CONHDoxy), 2.56 (dd, 1H, J = 12.4, 8.3 Hz, H-5a), 2.75 (br t, 3H J = 7.4 Hz, CH2-C-N
and H-6), 2.82 (d, 1H, J = 11.3, H-4a), 2.96 (br s, 6H, N(CH3)2), 3.56 (dd, 1H, J = 11.7, 8.3 Hz, H-5),
3.70 (s, 3H, CH3 methyl esther), 3.89 (s, 2H, CH2 glycine), 4.42 (s, 1H, H-4), 4.43 (t, 2H, J = 6.9 Hz,
CH2N-N), 6.93 (d, 1H, J = 8.3 Hz, H-7), 7.81 (s, 1H, H-= triazole), 8.11 (d, 1H, J = 8.3 Hz, H-8).
Interpretation is partly based on the following 2D spectra.
Experimental Part
177
76. H-H COSY
(S)-Methyl 2-[4-[1-[5-(doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-triazol-4-yl] butanamido]-3-phenylpropanoate (77)
The reaction followed the general procedure, starting from 10 milligrams (0.12 mmol) of derivative 72.
Characterization Yield : 6.1 mg (60%) as yellow film Analytical Data : C43H51N7O12 (MW = 857,93) APCI-MS m/z = 858.4 [M+1]+
Experimental Part
178
HPLC : tr = 17.1 min. purity > 99 % (254nm) IR (film) : 3600-3160 (OH), 3063 (Aromatic H), 2977, 2955, 2874 (alkyl chain), 1732,
1670, 1615, 1529 (C=O, Amide and C=C), 1428, 1241, 1199, 1045 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.53 (d, 3H, J = 6.4 Hz, CH3 at C-6), 1.69 (tt, 2H, J = 7.0, 7.0 Hz, CH2CH2COPhe), 1.82-
1.89 (m, 2H, CH2CH2N-N), 2.00 (tt, 2H, J = 7.4, 7.4 Hz, CH2CH2CONHDoxy), 2.20 (t, 2H, CH2-
CONHPhe), 2.51 (t, 2H, J = 6.8 Hz, CH2CONHDoxy), 2.56 (dd, 1H, J = 12.5, 8.5 Hz, H-5a), 2.56-2.62
(m, 2H, CH2-C-N), 2.70-2.76 (m, 1H, H-6), 2.81 (d, 1H, J = 11.3 Hz, H-4a), 2.98 (br s, 6H, N(CH3)2),
2.91 and 3.13 (2 dd,each 1H, J = 14.0, 9.4 Hz, J = 14.0, 5.8 Hz, diasterotopic CH2 Phe), 3.56 (dd, 1H,
J = 11.0, 8.3 Hz, H-5), 3.68 (s, 3H, CH3 methyl esther), 4.40-4.44 (m, 3H, CH2N-N, H-4), 4.67 (dd, 1H,
J = 9.4, 5.8 Hz, H alpha Phe), 6.93 (d, 1H, J = 8.3 Hz, H-7), 7.15-7.27 (m, 5H, Phenyl), 7.72 (s, 1H, H-
= triazole), 8.12 (d, 1H, J = 8.3 Hz, H-8).
13C NMR (90 MHz, CD3OD) :
δ (ppm) = 16.15 (CH3), 17.36, 18.79, 23.70 (CH2Phe, CH2CH2CO-Phe, CH2C-N), 26.42, 30.69
(CH2CH2N, C-4a), 35.41, 35.78, 36.76, (CHCOOH, CH2CONH, CH2CONHDoxy), 38.42 (C-6), 39.85
(N(CH3)2), 43.88 (C-5a), 51.10 (CH2-N-N), 52.73 (OCH3), 55.17 (CH alpha Phe), 67.38 (C-4), 70.08
(C-5), 74.68 (C-12a), 96.17 (C-2), 108.58 (C-11a), 116.21, 116.98 (C-7, C-10a), 123.60 (H-C=
triazole), 126.61, 127.91, 129.55, 130.23, 130.40, 138.34 (C-9, C-8, Phenyl Cs, N-C=CH), 144.65 (C-
6a), 154.23 (C-10), 172.94, 173.67, 174.25, 175.49 (C-12, CONH2, COOMe, CONHDoxy), 195.60,
195.64 (C1, C3, C11).
Experimental Part
179
General procedure for the synthesis of C-terminus modified amino acid building blocks: In 10 mL dry DMF were dissolved 4.4 mmol of N-Boc amino acid and EDC plus 6 mmol of TEA. The
solution was put in an ice bath and 4.4 mmol HOAt in 5 mL were added. After 5 minutes 2 mmol of 3-
amino-1-azide propane (synthesized following Hatzakis et al. Chem. Commun., 2006, pp. 2012-2014)
in mL of DMF were added. The reaction was sirred at 0°C for 30 minutes, then the ice bath was
removed and the reaction continued overnight.
To the solution were added 200 mL of a 10% water solution of citric acid and extracted 3x100mL with
ethylacetate.The organic phases were collected, washed 1x with 1N HCl, 1x with brine, 2x 1N NaOH,
dried over Na2SO4 and concentrated at rotatory evaporator. Purification by flash chromatography on
silica gel afforded pure amino acid derivative.
Eventual Boc deprotection was carried out dissolving the derivatives in 50% TFA in DCM and stirring
at room temperature for 1 hour, monitored by TLC analysis with ninhydrin detection. Solvent was
evaporated at rotavapor, 10 mL of saturated NaHCO3 were added and the solution extracted 3x with
ethylacetate. The organics were collected, dried over Na2SO4, evaporated in vacuo to afford the TFA
salt of the amino acid derivatives, which were used without further purifications.
N-(3-azidopropyl)-2-(dimethylamino) acetamide (78)
0.200 grams of 3-amino-1-azide propane was reacted with 453.7 mg of N,N-Dimethylglycine following
the general procedure. Purification by flash chromatography (EtOAc:n-Hexan 1:1) afforded the pure
compound.
Characterization Yield : 362 mg (98%) as yellow oil Analytical Data : C7H15N5O (MW = 185,23) IR (film) : 3350-3200, 2969, 2942, 2869, 2823, 2780, 2098, 1666, 1527, 1457, 1265,
1045 cm-1
HR-EIMS : Calculated: 185.1277
Measured: 185.1278
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.77 (dt, 2H, J = 6.7, 6.7 Hz, central CH2 linker), 2.29 (s, 6H, (CH3)2N), 2.96 (s, 2H, CH2
alpha), 3.39 and 3.35 (2 t, each 2H, J = 6.7 Hz, CH2NH and CH2-N3), 4.84 (s, 1H, NH).
13C NMR (90 MHz, CD3OH) :
Experimental Part
180
δ (ppm) = 29.77 (central CH2 linker), 37.51 (CH2NH), 45.98 ((CH3)2N), 51.21 (CH2N3), 63.61 (C alpha),
173.02 (CONH).
(S)-2-amino-N-(3-azidopropyl) propanamide (79)
0.200 grams of 3-amino-1-azide propane was reacted with 832 mg of N-Boc-alanine following the
general procedure. Purification by flash chromatography (EtOAc:n-Hexan 2:8) afforded the pure
compound, which was then Boc-deprotected following the general method.
Characterization of the Boc Derivative Yield : 420 mg (77%) as colorless oil Analytical Data : C11H21N5O3 (MW = 271,20) IR (film) : 3450-3150, 2978, 2933, 2874, 2509, 2096, 1681, 1651, 1455, 1368,
1248, 1167, 1056, 1020 cm-1
1H NMR (360 MHz, CDCl3) :
δ (ppm) = 1.30 (d, 3H, J = 7.0 Hz, CH3 Ala), 1.39 (s, 9H, 3 CH3 Boc), 1.69-1.78 (m, 2H, central CH2
linker), 3.20-3.34 (m, 4H, CH2NH and CH2-N3), 4.14 (br s, 1H, H alpha), 5.43-5.56 and 6.92-7.10 (2br
s, each 1H, NH amide). (spectra of the Boc-derivative)
13C NMR (90 MHz, CDCl3) :
δ (ppm) = 18.38 (CH3 Ala), 28.32, 28.55 (3 CH3 Boc, central CH2 linker), 36.60 (CH2NH), 48.85
(CH2N3), 49.95 (C alpha), 79.61 (C(CH3)3), 155.42 (C=O Boc), 173.10 (CONH).
(S)-2-amino-N-(3-azidopropyl)-3-phenylpropanamide (80)
Experimental Part
181
0.200 grams of 3-amino-1-azide propane was reacted with 1.167 g of N-Boc-phenylalanine following
general procedure. Purification by flash chromatography (EtOAc:n-Hexan 2:8) afforded the pure
compound, which was then Boc-deprotected following the general method.
Characterization of the Boc Derivative Yield : 695 mg (100%) as colorless oil Analytical Data : C17H25N5O3 (MW = 347,42) APCI-MS m/z = 348.2 [M+1]+
HR-ESI-MS Calculated: 347.1957
Measured: 347.1957
HPLC : tr = 18.2 min. purity 90 % (220nm) IR (film) : 3309, 3062, 3031, 2935, 2869, 2098, 1666, 1527, 1457, 1265, 1045 cm-1
1H NMR (360 MHz, CDCl3) :
δ (ppm) = 1.41 (s, 9H, 3 CH3 Boc), 1.63 (tt, 2H, J = 6.6, 6.6 Hz, central CH2 linker), 3.00-3.34 (m, 6H,
CH2-CONHPhe, CH2 Phe, CH2-N3), 4.27-4.36 (m, 1H, H alpha Phe), 5.20-5.26 and 6.20-6.30 (2 br s,
each 1H, 2 NH amides), 7.15-7.32 (m, 5H, aromatics).
13C NMR (90 MHz, CDCl3) :
δ (ppm) = 28.81, 28.48 (3 CH3 Boc, central CH2 linker), 36.75, 38.67 (CH2CONHPhe, CH2 Phe), 48.87
(CH2N3), 53.23 (C alpha), 80.12 (C(CH3)3), 126.87, 128.56, 129.22, 136.73 (Aromatics), 155.41 (C=O
Boc), 171.39 (CONH).
(S)-2-amino-N-(2-(3-azidopropylamino)-2-oxoethyl)propanamide (81)
0.200 grams of 3-amino-1-azide propane was reacted with 591 mg of N-Boc-Ala-Gly-OH following the
general procedure, but using 1.2 equivalents (2.4 mmol) of dipeptide, HOAt and EDC. Purification
through extractions gave the compound in sufficient purity and no purification via flash
chromatography was necessary. Before click reaction, 81 was Boc-deprotected following the general
method.
Characterization of the Boc Derivative Yield : 656 mg (100%) as light yellow oil Analytical Data : C13H24N6O4 (MW = 328,37)
Experimental Part
182
IR (film) : 3600-3200, 2946, 2877, 2827, 2780, 2098, 1666, 1527, 1457, 1268, 1045 cm-1 1H NMR (600 MHz, DMSO-d6) :
δ (ppm) = 1.17 (d, 3H, J = 7.2 Hz, CH3 Ala), 1.39 (s, 9H, 3 CH3 Boc), 1.65 (tt, 2H, J = 6.8, 6.8 Hz,
central CH2 linker), 3.05-3.20 (m, 2H, CH2-CONH), 3.34 (t, 2H, J = 6.8 Hz, CH2-N3), 3.61 and 3.67 (2
dd, each 1H, J = 16.4, 6.0 Hz, CH2 alpha gly), 3.90-3.96 (m, 1H, H alpha Ala), 6.11 (d, 1H, J = 7.5 Hz,
NH), 7.06-7.12, 7.67-7.74 and 8.06-8.12 (3 br s, each 1H, NHs).
13C NMR (90 MHz, DMSO-d6) :
δ (ppm) = 17.63 (CH3 Ala), 28.16, 28.34 (3 CH3 Boc, central CH2 linker), 35.79 (CH2CONH), 42.17 (C
alpha Gly), 48.25 (CH2N3), 50.01 (C alpha Ala), 78.27 (C(CH3)3), 155.41 (C=O Boc), 168.74, 173.02 (2
CONH).
9-[4-[1-[3-[2-(dimethylamino)acetamido]propyl]-1H-1,2,3-triazol-4-yl]butanamido] doxycycline (82)
Following procedure described at page 166, 25 mg of derivative 51 (MW = 553.57, 0.045 mmol), 13 eq
of CuI (0.58 mmol, 111 mg) and 7 eq of ascorbic acid (0.315 mmol, 55 mg) were solved in 2 mL of dry
DMF. To this solution were added 2 equivalents of 78 (MW = 185,23, 17 mg) in 1 mL of dry DMF, and
17 eq of DIPEA (0.765 mmol, 0.13 mL). Reaction and purification followed the general procedure.
Characterization Yield : 5.6 mg (17%) Analytical Data : C35H46N8O10 (MW = 738,80) APCI-MS m/z = 739.3 [M+1]+ HPLC : tr = 3.1 min. purity > 95 % (254nm) IR (film) : 3550-3200 (OH), 3047(Aromatic H), 2971, 2859 (alkyl chain), 1669, 1622,
1521 (C=O, Amide and C=C), 1471, 1430, 1204, 1129 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.57 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.12 (tt, 2H, J = 7.4, 7.4 Hz, CH2CH2CONHDoxy), 2.19
(tt, 2H, J = 6.9, 6.9 Hz, CH2CH2N-N), 2.58 (t, 2H, J = 7.4 Hz, CH2-CONHDoxy), 2.61 (dd, 1H, J = 12.3,
8.5 Hz, H-5a), 2.79 (dq, 1H, J = 6.6, 13.3 Hz, H-6), 2.85 (d, 1H, J = 11.3 Hz, H-4a), 2.91 (t, 2H, J = 7.6
Experimental Part
183
Hz, CH2C-N-N), 2.92-3.04 (m, 12H, CH2N(CH3)2 + N(CH3)2 doxy), 3.31 (t, 2H, CONH-CH2CH2, under
MeOD signal), 3.58 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 3.97 (s, 2H, CH2-N(CH3)2), 4.45 (s, 1H, H-4), 4.54
(t, 2H, J = 6.8 Hz, CH2N-N), 6.97 (d, 1H, J = 8.3 Hz, H-7), 8.14 (s, 1H, H-= triazole), 8.16 (d, 1H, J =
8.3 Hz, H-8).
9-[4-[1-[3-((S)-2-aminopropanamido)propyl]-1H-1,2,3-triazol-4-yl]butanamido] doxycycline (83)
Following procedure described at page 166, 20 mg of derivative 51 (MW = 553.57, 0.036 mmol), 13 eq
of CuI (0.47 mmol, 89 mg) and 7 eq of ascorbic acid (0.25 mmol, 44 mg) were solved in 2 mL of dry
DMF. To this solution were added 2 equivalents of 79 (MW = 171.32, 12 mg) in 1 mL of dry DMF, and
17 eq of DIPEA (0.614 mmol, 0.1 mL). Reaction and purification followed the general procedure.
Characterization Yield : 8.1 mg (30.8%) Analytical Data : C34H44N8O10 (MW = 724,78) APCI-MS m/z = 725.4 [M+1]+ HPLC : tr = 12.5 min. purity > 95 % (254nm) IR (film) : 3500-3190 (OH), 3052 (Aromatic H), 2971, 2920, 2859 (alkyl chain), 1673, 1622,
1523 (C=O, Amide and C=C), 1471, 1430, 1202, 1135 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.50 (d, 3H, J = 7.0 Hz, CH3 Ala), 1.55 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.07 (tt, 2H, J = 7.0,
7.1 Hz, CH2CH2CONHDoxy), 2.13 (tt, 2H, J = 7.0, 7.0 Hz, CH2CH2N-N), 2.53 (t, 2H, J = 7.4 Hz, CH2-
CONHDoxy), 2.60 (dd, 1H, J = 12.1, 8.3 Hz, H-5a), 2.76 (dq, 1H, J = 6.6, 13.6 Hz, H-6), 2.82 (t, 2H, J
= 7.4 Hz, CH2C-N-N), 2.84 (d, 1H, J = 11.7 Hz, H-4a), 2.91-3.02 (br s, 6H, N(CH3)2), 3.20-3.27 (m,
2H, CONH-CH2CH2), 3.57 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 3.91 (q, 1H, J = 7.1 Hz, H alpha Ala), 4.43
(t, 2H, J = 7.0 Hz, CH2N-N), 4.44 (s, 1H, H-4), 6.95 (d, 1H, J = 8.3 Hz, H-7), 7.86 (s, 1H, H-= triazole),
8.15 (d, 1H, J = 8.3 Hz, H-8).
Experimental Part
184
9-[4-[1-[3-((S)-2-amino-3-phenylpropanamido)propyl]-1H-1,2,3-triazol-4-yl)butanamido] doxycycline (84)
Following procedure described at page 166, 20 mg of derivative 51 (MW = 553.57, 0.036 mmol), 13 eq
of CuI (0.47 mmol, 89 mg) and 7 eq of ascorbic acid (0.25 mmol, 44 mg) were solved in 2 mL of dry
DMF. To this solution were added 2 equivalents of 80 (MW = 247.22, 18 mg) in 1 mL of dry DMF, and
17 eq of DIPEA (0.614 mmol, 0.1 mL). Reaction and purification followed the general procedure.
Characterization Yield : 15.4 mg (53.3%) Analytical Data : C40H48N8O10 (MW = 800,88) APCI-MS m/z = 802.3 [M+1]+ HPLC : tr = 14.0 min. purity > 95 % (254nm) IR (film) : 3500-3200 (OH), 3093 (Aromatic H), 2992, 2873 (alkyl chain), 1781, 1670,
1535 (C=O, Amide and C=C), 1430, 1168, 1037, 701 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.55 (d, 3H, J = 6.8 Hz, CH3 at C-6), 1.98 (tt, 2H, J = 6.5, 6.4 Hz, CH2CH2CONHDoxy),
2.07 (tt, 2H, J = 7.3, 7.1 Hz, CH2CH2N-N), 2.53 (t, 2H, J = 7.2 Hz, CH2-CONHDoxy), 2.59 (dd, 1H, J =
12.4, 8.4 Hz, H-5a), 2.72-2.77 (m, 1H, H-6), 2.78-2.84 (m, 3H, CH2C-N-N, H-4a), 2.96 (br s, 6H,
N(CH3)2), 3.07-3.25 (m, 4H, CONH-CH2CH2, CH2-Phe), 3.57 (dd, 1H, J = 10.7, 8.2 Hz, H-5), 4.04 (t,
1H, J = 7.6 Hz, H alpha Phe), 4.19-4.28 (m, 2H, CH2N-N), 4.43 (s, 1H, H-4), 6.94 (d, 1H, J = 8.2 Hz,
H-7), 7.26-7.37 (m, 5H, Phenyl), 7.78 (s, 1H, H-= triazole), 8.15 (d, 1H, J = 8.2 Hz, H-8).
Experimental Part
185
9-[4-[1-[3-[2-((S)-2-aminopropanamido)acetamido]propyl]-1H-1,2,3-triazol-4-yl]butanamido] doxycycline (85)
Following procedure described at page 166, 20 mg of derivative 51 (MW = 553.57, 0.036 mmol), 13 eq
of CuI (0.47 mmol, 89 mg) and 7 eq of ascorbic acid (0.25 mmol, 44 mg) were solved in 2 mL of dry
DMF. To this solution were added 2 equivalents of 81 (MW = 228,32, 16 mg) in 1 mL of dry DMF, and
17 eq of DIPEA (0.614 mmol, 0.1 mL). Reaction and purification followed the general procedure.
Characterization Yield : 6.2 mg (22%) as yellow glas Analytical Data : C36H47N9O11 (MW = 781,83) APCI-MS m/z = 782.4 [M+1]+ HPLC : tr = 12.5 min. purity > 95 % (254nm) IR (film) : 3081 (Aromatic H), 2981, 2892 (alkyl chain), 1785, 1650, 1419, 1234, 1172, 1037,
705 cm-1
1H NMR (600 MHz, CD3OD) :
δ (ppm) = 1.52 (d, 3H, J = 7.2 Hz, CH3 Ala), 1.55 (d, 3H, J = 6.8 Hz, CH3 at C-6), 2.05-2.15 (m, 4H,
CH2CH2CONHDoxy, CH2CH2N-N), 2.54 (t, 2H, J = 7.2 Hz, CH2-CONHDoxy), 2.59 (dd, 1H, J = 12.1,
8.3 Hz, H-5a), 2.76 (dq, 1H, J = 6.2, 12.5 Hz, H-6), 2.81-2.86 (m, 3H, H-4 + CH2C-N-N), 2.91-3.00 (br
s, 6H, N(CH3)2), 3.24 (t, 2H J = 6.6 Hz, CONH-CH2CH2), 3.57 (dd, 1H, J = 11.3, 8.3 Hz, H-5), 3.84 and
3.94 (2 d, each 1 H, J = 16.6 Hz, H alpha Gly), 4.00 (q, 1H, J = 7.1 Hz, H alpha Ala), 4.43 (s, 1H, H-4),
4.44 (t, 2H, J = 7.0 Hz, CH2N-N), 6.95 (d, 1H, J = 8.3 Hz, H-7), 7.90 (s, 1H, H-= triazole), 8.15 (d, 1H, J
= 8.3 Hz, H-8).
Experimental Part
186
General procedure for the synthesis of peptide conjugates :
Peptides were synthesized by Dr. Jürgen Einsiedel with microwave assisted Fmoc-SPPS and used for
the conjugation in their totally unprotected, crude form.
In a microwave vial 1 equivalent of derivative 51 or 53, 13 eq of CuI, 7 eq of ascorbic acid plus 1.2-2
equivalents (according to purity) of peptide were dissolved in a 1:1mixture (2 mL) of dry DMF and dry
acetonitrile. The vial was sealed, degassed and charged with nitrogen. Finally, 17 eq of DIPEA were
added via syringe. After a 2 min treatment in a bath sonicator, the reaction mixture was heated to
50°C in oil bath. Following LC-MS analysis, the reactions were complete within 1-2 hour. The mixture
was diluted with 2 mL H2O, filtered or centrifugued and the solution poured in 100 mL of 1N HCl, then
freezed and lyophilized. The crude product was dissolved in a mixture of DMF/MeOH (1:3) acidic per
HCl and purified through RP-HPLC, obtaining pure peptide conjugates.
86
10 mg of derivative 51 (MW = 553.57, 0.018 mmol), 13 eq of CuI (0.23 mmol, 45 mg), 7 eq of ascorbic
acid (0.126 mmol, 22 mg) and 1.5 equivalents of peptide N3(CH2)4CO-DFDLDMLG-NH2 (MW =
1049.18, mmol 0.027, 28.4 mg) were solved in 2 mL of dry DMF/CH3CN (1:1). To this solution were
added 17 eq of DIPEA (0.307 mmol, 0.05 mL) and the general procedure followed. The reaction was
complete within 1 hour.
Characterization Yield : 8 mg (28.6%) as TFA salt Analytical Data : C73H99N15O24S (MW = 1602,75) ESI-MS Calculated: m/z = 1602.7 [M+1]+ --- 801.8 [M+2]+/2 Measured: m/z = 1602.8 [M+1]+ --- 802.1 [M+2]+/2
Experimental Part
187
HPLC : tr = 11.3 min. purity > 95 % (254nm) 1H NMR (600 MHz, Pyr-d5) :
δ (ppm) = 0.85 (d, 6H, J=6.4 Hz, CH3 Leu), 0.88 (d, 3H, J=6.4 Hz, CH3 Leu), 0.92 (d, 3H, J=6.4 Hz,
CH3 Leu), 1.68-1.78 (m, 2H, CH2CH2COAsp1), 1.74 (d, 3H, J = 6.3 Hz, CH3 at C-6), 1.86-1.98 (m, 4H,
CβH2 Leu, CH2CH2N-N), 1.98-2.15 (m, 4H, cβH2Leu, CγH2 Leu), 1.99 (s, 3H, SCH3), 2.30-2.40 (m, 4H,
H-5a, H-6, CH2CH2CONHDoxy), 2.45-2.53 (m, 1H, CβH2 Met), 2.55-2.63 (m, 1H, CβH2 Met), 2.67 (br s,
6H, N(CH3)2), 2.77 (t, 2H, J = 7.2 Hz, CH2-COAsp1), 2.78-2.84 (m, 1H, CγH2 Met), 2.87-2.94 (m, 2H,
CγH2 Met, H-4a), 2.96 (t, 2H, J = 7.55 Hz, CH2CONHDoxy), 3.08-3.14 (m, 2H, CH2-C-N), 3.10 (dd, 1H,
J = 6.2, 16.4 Hz, CβH2 Asp1),3.21 (dd, 1H, J = 7.2, 16.6 Hz, CβH2 Asp2), 3.30 (dd, 1H, J = 9.2, 14.2
Hz, CβH2Phe), 3.34 (2 dd, 2H, J = 8.5, 16.4 Hz and 7.4, 16.4 Hz, CβH2 Asp3 and CβH2 Asp2), 3.40-
3.47 (m, 2H, CβH2 Asp3 and CβH2 Asp1), 3.53 (dd, 1H, J= 14.0, 4.9 Hz, CβH2Phe), 3.88 (br d, 1H, J =
9.06 Hz, H-5), 4.31 (t, 2H, J = 7.0 Hz, CH2N-N) 4.34 (dd, 1H, J = 5.9, 16.8 Hz, CαH2Gly), 4.45 (dd, 1H,
J = 6.4, 16.9 Hz, CαH2Gly), 4.60 (s, 1H, H-4), 4.79, 4.96 and 5.04 (3 m, each 1H, CαH Leu, Leu and
Met), 5.18 (m, 1H, CαHAsp3), 5.39, 5.44 and 5.48 (3 dt, each 1H, J = 6.8, 7.0 Hz; J = 7.2, 7.2 Hz; J =
7.3, 7.4 Hz; CαH Asp2, Phe, Asp1), 6.98 (d, 1H, J = 8.3 Hz, H-7), 7.20 (t, 1H, J = 7.4 Hz, H-4’ Phe),
7.27 (dd, 2H, J = 7.55, 7.55 Hz, H-3’ and H-5’ Phe), 7.37 (t, 2H, J = 7.2 Hz, H-2’ and H-6’ Phe), 7.58
(s, 1H, H at C-5 triazole), 8.05 and 8.16 (2 s, each 1H, CONH2 C-Terminus), 8.62 (d, 1H, J = 7.1Hz,
NH), 8.72 (d, 1H, J = 7.1 Hz, NH), 8.80 (d, 1H, J = 8.3 Hz, H-8), 8.91 (dd, 1H, J = 5.9, 6.1 Hz, NH Gly),
8.97 (d, 1H, J = 6.8 Hz, NH), 9.11 (d, 1H, J = 6.4 Hz, NH), 9.32 (d, 1H, J = 7.6 Hz, NH), 9.36 (d, 1H, J
= 7.6 Hz, NH), 9.58 (d, 1H, J = 6.8 Hz, NH), 9.76 (s, 1H, NH at C-9), 10.07 (br s, 1H, CONH2 Doxy),
10.26 (br s, 1H, CONH2 Doxy).
87
Experimental Part
188
10 mg of derivative 51 (MW = 553.57, 0.018 mmol), 13 eq of CuI (0.23 mmol, 45 mg), 7 eq of ascorbic
acid (0.126 mmol, 22 mg) and 1 equivalent of peptide N3(CH2)4CO-DFDLDMLG-
(CH2CH2O)2CH2CONH2 (MW = 1138.39, mmol 0.018, 22.4 mg) were solved in 2 mL of dry
DMF/CH3CN (1:1). To this solution were added 17 eq of DIPEA (0.307 mmol, 0.05 mL) and the
general procedure followed. The reaction was complete within 1 hour.
Characterization Yield : 15.3 mg (47.3%) as TFA salt Analytical Data : C81H114N16O28S (MW = 1791,96) ESI-MS Calculated: m/z = 1791.7 [M+1]+ --- 896.9 [M+2]+/2 --- 597.9 [M+3]+/3 Measured: m/z = 1791.7 [M+1]+ --- 896.8 [M+2]+/2 --- 598.1 [M+3]+/3
HPLC : tr = 11.4 min. purity > 99 % (254nm) IR (film) : 3300-2560 (OH), 3088 (Aromatic H), 2943, 2933 (alkyl chain), 2871 (OCH2,
SCH3), 1750-1600 (C=O), 1595 (C=C), 1537 (C=C) cm-1
1H NMR (600 MHz, Pyr-d5) :
δ (ppm) = 0.94 (d, 6H, J=6.9 Hz, CH3 Leu), 0.95 (d, 3H, J=6.8 Hz, CH3 Leu), 1.00 (d, 3H, J=6.0 Hz,
CH3 Leu), 1.75-1.85 (m, 2H, CH2CH2COAsp1), 1.81 (d, 3H, J = 6.4 Hz, CH3 at C-6), 1.97 (tt, 2H, J =
7.4, 7.5 Hz, CH2CH2N-N), 2.00-2.06 (m, 2H, CβH2 Leu), 2.07-2.15 (m, 4H, cβH2Leu, CγH2 Leu), 2.09 (s,
3H, SCH3), 2.35-2.48 (m, 4H, H-5a, H-6, CH2CH2CONHDoxy), 2.53-2.61 (m, 1H, CβH2 Met), 2.64-2.71
(m, 1H, CβH2 Met), 2.73 (br s, 6H, N(CH3)2), 2.85 (t, 2H, J = 7.4 Hz, CH2-COAsp1), 2.86-2.92 (m, 1H,
CγH2 Met), 2.96-3.02 (m, 2H, CγH2 Met, H-4a), 3.03 (t, 2H, J = 7.55 Hz, CH2CONHDoxy), 3.14-3.22
(m, 2H, CH2-C-N), 3.20 (dd, 1H, J = 4.1 13.2 Hz, CβH2 Asp1), 3.30 (dd, 1H, J = 7.2, 16.6 Hz, CβH2
Asp2), 3.37 (dd, 1H, J = 9.1, 14.0 Hz, CβH2Phe), 3.41 and 3.42 (2 dd, 2H, J = 8.3, 16.6 Hz and 7.4,
16.4 Hz, CβH2 Asp3 and CβH2 Asp2), 3.47-3.53 (m, 2H, CβH2 Asp3 and CβH2 Asp1), 3.60 (dd, 1H, J=
14.2, 5.1 Hz, CβH2Phe), 3.65-3.70 (m, 6H, (OCH2CH2)3), 3.73-3.77 (m, 6H, (OCH2CH2)3), 3.95 (br d,
1H, J = 9.4 Hz, H-5), 4.30 (s, 2H, H2NCOCH2O), 4.38 (t, 2H, J = 7.4 Hz, CH2N-N), 4. 43 (dd, 2H, J =
6.0, 16.6 Hz, CαH2Gly), 4.68 (br s, 1H, H-4), 4.86, 5.03 and 5.13 (3 m, each 1H, CαH Leu, Leu and
Met), 5.24 (m, 1H, CαHAsp3), 5.44, 5.50 and 5.56 (3 dt, each 1H, J = 6.6, 7.1 Hz; J = 7.2, 7.2 Hz; J =
7.2, 7.4 Hz; CαH Asp2, Phe, Asp1), 7.05 (d, 1H, J = 8.7 Hz, H-7), 7.27 (t, 1H, J = 7.2 Hz, H-4’ Phe),
7.34 (dd, 2H, J = 7.55, 7.55 Hz, H-3’ and H-5’ Phe), 7.45 (d, 2H, J = 7.5 Hz, H-2’ and H-6’ Phe), 7.65
(s, 1H, H at C-5 triazole), 7.94 (br s, 1H, CONH2 mini-Peg), 8.31 (br s,1H, CONH2 mini-Peg), 8.39 (m,
1H, CONH mini-Peg), 8.63 (d, 1H, J = 7.2Hz, NH), 8.78 (d, 1H, J = 7.2 Hz, NH), 8.88 (d, 1H, J = 8.8
Hz, H-8), 8.90 (d, 1H, J = 6.0 Hz, NH Gly), 9.00 (d, 1H, J = 6.8 Hz, NH), 9.16 (d, 1H, J = 6.4 Hz, NH),
9.40 (d, 1H, J = 7.2 Hz, NH), 9.42 (d, 1H, J = 8.0 Hz, NH), 9.64 (d, 1H, J = 6.8 Hz, NH), 9.82 (s, 1H,
NH at C-9), 10.15 (br s, 1H, CONH2 Doxy), 10.37 (br s, 1H, CONH2 Doxy).
Experimental Part
189
88
10 mg of derivative 51 (MW = 553.57, 0.018 mmol), 13 eq of CuI (0.23 mmol, 45 mg), 7 eq of ascorbic
acid (0.126 mmol, 22 mg) and 1.5 equivalents of peptide N3(CH2)4CO-SMWRPWRNG-NH2 (MW =
1313.49, mmol 0.027, 35.5 mg) were solved in 2 mL of dry DMF/CH3CN (1:1). To this solution were
added 17 eq of DIPEA (0.307 mmol, 0.05 mL). Following general procedure, the reaction was
complete within 2 hour.
Characterization Yield : 15.1 mg (45%) as TFA salt Analytical Data : C86H115N25O21S (MW = 1867,09) ESI-MS Calculated: m/z = 1866.85 [M+1]+ --- 933.93 [M+2]+/2 --- 622.95 [M+3]+/3 Measured: m/z = 1867.8 [M+2]+ --- 934.1 [M+2]+/2 --- 623.3 [M+3]+/3
HPLC : tr = 3.9 min. purity > 99 % (220nm) double peak
Experimental Part
190
89
10 mg of derivative 53 (MW = 584.59, 0.017 mmol), 13 eq of CuI (0.22 mmol, 42 mg), 7 eq of ascorbic
acid (0.12 mmol, 21 mg) and 1.5 equivalents of peptide HC≡C(CH2)3CO-DFDLDMLG-NH2 (0.026
mmol, 26 mg) were dissolved in 2 mL of dry DMF/CH3CN (1:1). To this solution were added 17 eq of
DIPEA (0.29 mmol, 0.05 mL). Following general procedure, the reaction was complete within 1 hour.
Characterization Yield : 16.8 mg (62%) as TFA salt Analytical Data : C73H99N15O24S (MW = 1602,75) ESI-MS Calculated: m/z = 1602.7 [M+1]+ --- 801.8 [M+2]+/2 Measured: m/z = 1602.8 [M+1]+ --- 802.1 [M+2]+/2
HPLC : tr = 12.1 min. purity > 95 % (254nm) IR (film) : 3400-2620 (OH), 3081 (Aromatic H), 2956, 2943 (alkyl chain), 2871 (OCH2,
SCH3), 1750-1600 (C=O), 1589 (C=C), 1528 (C=C) cm-1
1H NMR (600 MHz, Pyr-d5) :
δ (ppm) = 0.85 (d, 6H, J=6.4 Hz, CH3 Leu), 0.89 (d, 3H, J=6.0 Hz, CH3 Leu), 0.93 (d, 3H, J=6.4 Hz,
CH3 Leu), 1.74 (d, 3H, J = 6.0 Hz, CH3 at C-6), 1.83 (tt, 2H, J = 7.3, 7.4 Hz, CH2CH2CONHDoxy),
1.87-2.16 (m and s (2.00 ppm), 11H, CβH2 Leu, CH2CH2CONHAsp1, cβH2Leu, CγH2 Leu, SCH3), 2.20
(m, 2H, CH2CH2N-N), 2.37-2.55 (m, 3H, H-5a, H-6, CβH2 Met), 2.56-2.63 (m, 1H, CβH2 Met), 2.65 (t,
2H, J = 7.2 Hz, CH2-CONHAsp1), 2.69 (br s, 6H, N(CH3)2), 2.80 (ddd, 1H, J= 13.3, 7.1, 6.1 Hz, CγH2
Met), 2.86-2.98 (m, 4H, CγH2 Met, H-4a, CH2CONHDoxy), 3.06-3.15 (m, 3H, CH2-C-N, CβH2 Asp1),
3.24 (dd, 1H, J = 7.2, 16.6 Hz, CβH2 Asp2), 3.28-3.47 (m, 5H, CβH2Phe, CβH2 Asp3 and CβH2 Asp2,
CβH2 Asp3 and CβH2 Asp1), 3.53 (dd, 1H, J= 14.0, 4.9 Hz, CβH2Phe), 3.92 (br d, 1H, J = 8.3 Hz, H-5),
4.33 (dd, 1H, J = 6.2, 16.8 Hz, CαH2Gly), 4.35 (t, 2H, J = 7.0 Hz, CH2N-N), 4.45 (dd, 1H, J = 6.3, 16.8
Experimental Part
191
Hz, CαH2Gly), 4.59 (s, 1H, H-4), 4.77, 4.96 and 5.03 (3 m, each 1H, CαH Leu, Leu and Met), 5.17 (m,
1H, CαHAsp3), 5.37, 5.42 and 5.48 (3 dt, each 1H, J = 6.8, 7.0 Hz; J = 7.0, 7.2 Hz; J = 7.0, 7.0 Hz;
CαH Asp2, Phe, Asp1), 6.98 (d, 1H, J = 8.3 Hz, H-7), 7.18 (t, 1H, J = 7.2 Hz, H-4’ Phe), 7.26 (dd, 2H, J
= 7.55, 7.55 Hz, H-3’ and H-5’ Phe), 7.39 (d, 2H, J = 7.2 Hz, H-2’ and H-6’ Phe), 7.58 (s, 1H, H at C-5
triazole), 8.04 and 8.16 (2 s, each 1H, CONH2 C-Terminus), 8.58 (d, 1H, J = 7.2Hz, NH), 8.68 (d, 1H,
J = 6.8 Hz, NH), 8.77 (d, 1H, J = 8.3 Hz, H-8), 8.88 (dd, 1H, J = 5.9, 6.1 Hz, NH Gly), 8.93 (d, 1H, J =
6.4 Hz, NH), 9.06 (d, 1H, J = 6.0 Hz, NH), 9.30 (d, 1H, J = 7.2 Hz, NH), 9.39 (d, 1H, J = 7.2 Hz, NH),
9.61 (d, 1H, J = 6.8 Hz, NH), 9.73 (s, 1H, NH at C-9), 10.06 (br s, 1H, CONH2 Doxy), 10.25 (br s, 1H,
CONH2 Doxy).
90
5 mg of derivative 53 (MW = 584.59, 0.0085 mmol), 13 eq of CuI (0.11 mmol, 21 mg), 7 eq of ascorbic
acid (0.059 mmol, 10 mg) and 1 equivalent of peptide HC≡C(CH2)3CO-DFDLDMLG-
(CH2CH2O)2CH2CONH2 (0.0085 mmol, 10.2 mg) were dissolved in 2 mL of dry DMF/CH3CN (1:1). To
this solution were added 17 eq of DIPEA (0.145 mmol, 0.03 mL). Following general procedure, the
reaction was complete within 1 hour.
Characterization Yield : 6 mg (39.2%) as TFA salt Analytical Data : C81H114N16O28S (MW = 1791,96) ESI-MS Calculated: m/z = 1791.7 [M+1]+ --- 896.9 [M+2]+/2 --- 597.9 [M+3]+/3 Measured: m/z = 1791.8 [M+1]+ --- 896.8 [M+2]+/2 --- 598.2 [M+3]+/3
Experimental Part
192
HPLC : tr = 11.5 min. purity > 99 % (220nm) IR (film) : 3530-2900 (OH), 3103 (NH), 3079 (Aromatic H), 2956, 2922 (alkyl chain),
2871 (OCH2, SCH3), 1750-1640 (C=O), 1599 (C=C) cm-1
1H NMR (600 MHz, Pyr-d5) :
δ (ppm) = 0.85-0.90 (m, 3H, CH3 Leu, CH3 Leu), 0.93 (d, 3H, J=6.0 Hz, CH3 Leu), 1.74 (d, 3H, J = 6.4
Hz, CH3 at C-6), 1.83 (tt, 2H, J = 7.5, 7.6 Hz, CH2CH2CONHDoxy), 1.87-2.14 (m and s (2.02 ppm),
11H, CβH2 Leu, CH2CH2CONHAsp1, cβH2Leu, CγH2 Leu, SCH3), 2.20 (m, 2H, CH2CH2N-N), 2.37-2.55
(m, 3H, H-5a, H-6, CβH2 Met), 2.56-2.63 (m, 1H, CβH2 Met), 2.65 (t, 2H, J = 7.2 Hz, CH2-CONHAsp1),
2.69 (br s, 6H, N(CH3)2), 2.80 (ddd, 1H, J= 13.2, 7.9, 6.1 Hz, CγH2 Met), 2.86-3.00 (m, 4H, CγH2 Met,
H-4a, CH2CONHDoxy), 3.06-3.15 (m, 3H, CH2-C-N, CβH2 Asp1), 3.26 (dd, 1H, J = 7.4, 16.4 Hz, CβH2
Asp2), 3.28-3.47 (m, 5H, CβH2Phe, CβH2 Asp3 and CβH2 Asp2, CβH2 Asp3 and CβH2 Asp1), 3.53 (dd,
1H, J= 14.2, 5.1 Hz, CβH2Phe), 3.58-3.63 (m, 6H, (OCH2CH2)3), 3.65-3.70 (m, 6H, (OCH2CH2)3), 3.92
(br d, 1H, J = 7.6 Hz, H-5), 4.23 (s, 2H, H2NCOCH2O), 4.30-4.38 (m, 3H, CαH2Gly, CH2N-N), 4.59 (s,
1H, H-4), 4.77, 4.97 and 5.05 (3 m, each 1H, CαH Leu, Leu and Met), 5.17 (m, 1H, CαHAsp3), 5.36,
5.42 and 5.48 (3 dt, each 1H, J = 6.8, 7.0 Hz; J = 7.0, 7.1 Hz; J = 7.2, 7.2 Hz; CαH Asp2, Phe, Asp1),
6.98 (d, 1H, J = 8.3 Hz, H-7), 7.18 (t, 1H, J = 7.4 Hz, H-4’ Phe), 7.27 (dd, 2H, J = 7.55, 7.55 Hz, H-3’
and H-5’ Phe), 7.40 (d, 2H, J = 7.55 Hz, H-2’ and H-6’ Phe), 7.58 (s, 1H, H at C-5 triazole), 7.90 (br s,
1H, CONH2 mini-Peg), 8.25 (br s, 1H, CONH2 mini-Peg), 8.34 (m, 1H, CONH mini-Peg), 8.58 (d, 1H, J
= 7.2Hz, NH), 8.68 (d, 1H, J = 6.8 Hz, NH), 8.77 (d, 1H, J = 8.3 Hz, H-8), 8.82 (dd, 1H, J = 5.7, 5.9 Hz,
NH Gly), 8.90 (d, 1H, J = 6.8 Hz, NH), 9.05 (d, 1H, J = 6.0 Hz, NH), 9.30 (d, 1H, J = 7.2 Hz, NH), 9.39
(d, 1H, J = 7.55 Hz, NH), 9.61 (d, 1H, J = 6.8 Hz, NH), 9.72 (s, 1H, NH at C-9), 10.06 (br s, 1H,
CONH2 Doxy), 10.17 (br s, 1H, CONH2 Doxy).
Experimental Part
193
91
10 mg of derivative 53 (MW = 584.59, 0.017 mmol), 13 eq of CuI (0.22 mmol, 42 mg), 7 eq of ascorbic
acid (0.12 mmol, 21 mg) and 1.5 equivalents of peptide HC≡C(CH2)3CO- SMWRPWRNG-NH2 (0.026
mmol, 33 mg) were dissolved in 2 mL of dry DMF/CH3CN (1:1). To this solution were added 17 eq of
DIPEA (0.29 mmol, 0.05 mL). Following general procedure, the reaction was complete within 1 hour.
Characterization Yield : 18.6 mg (58.3%) as TFA salt Analytical Data : C86H115N25O21S (MW = 1867,09) ESI-MS Calculated: m/z = 1866.85 [M+1]+ --- 933.93 [M+2]+/2 --- 622.95 [M+3]+/3 Measured: m/z = 1867.8 [M+2]+ --- 934.1 [M+2]+/2 --- 623.3 [M+3]+/3
HPLC : tr = 3.9 min. purity > 99 % (220nm) double peak IR (film) : 3530-2400 (OH), 3079 (Aromatic H), 2956, 2925 (alkyl chain), 2871 (OCH2,
SCH3), 1750-1600 (C=O), 1595 (C=C), 1537 (C=C) cm-1
Experimental Part
194
92
5 mg of derivative 53 (MW = 584.59, 0.0085 mmol), 13 eq of CuI (0.11 mmol, 21 mg), 7 eq of ascorbic
acid (0.059 mmol, 10 mg) and 2 equivalent of peptide DFDLDMLG-(α-propargyl)G-NH2 (0.017 mmol,
17.4 mg) were dissolved in 2 mL of dry DMF/CH3CN (1:1). To this solution were added 17 eq of
DIPEA (0.145 mmol, 0.03 mL). Following general procedure, the reaction was complete within 1 hour.
Characterization Yield : 8 mg (58%) as TFA salt Analytical Data : C72H98N16O24S (MW = 1603,71) ESI-MS Calculated: m/z = 1603.67 [M+1]+ --- 802.34 [M+2]+/2 --- 535.23 [M+3]+/3 Measured: m/z = 1603.7 [M+1]+ --- 802.5 [M+2]+/2 --- 535.4 [M+3]+/3
HPLC : tr = 10.9 min. purity > 99 % (220nm) IR (film) : 3530-2530 (OH), 3079 (Aromatic H), 2956, 2943 (alkyl chain), 2871 (OCH2,
SCH3), 1750-1600 (C=O), 1595 (C=C), 1537 (C=C) cm-1
Experimental Part
195
93
10 mg of derivative 51 (MW = 553.57, 0.018 mmol), 13 eq of CuI (0.23 mmol, 45 mg), 7 eq of ascorbic
acid (0.126 mmol, 22 mg) and 1 equivalent of peptoide DFDLDMLG-N-[(CH2)3N3],N-CH2CONH2 (MW
= 1064.2, mmol 0.018, 19.2 mg) were solved in 2 mL of dry DMF/CH3CN (1:1). To this solution were
added 17 eq of DIPEA (0.307 mmol, 0.05 mL) and the general procedure followed. The reaction was
complete within 1 hour.
Characterization Yield : 12 mg (41%) as TFA salt Analytical Data : C73H100N16O24S (MW = 11617,76) ESI-MS Calculated: m/z = 1618.76 [M+1]+ --- 809.35 [M+2]+/2 --- 539.90 [M+3]+/3 Measured: m/z = 1618.6 [M+1]+ --- 809.4 [M+2]+/2 --- 540.1 [M+3]+/3
HPLC : tr = 11.0 min. purity > 99 % (220nm) IR (film) : 3600-2500 (OH), 3081 (Aromatic H), 2956, 2926 (alkyl chain), 2871 (OCH2,
SCH3), 1750-1600 (C=O), 1595 (C=C), 1537 (C=C) cm-1
Abbreviations and Acronyms
196
Abbreviations and Acronyms APCI atmospheric pressure chemical ionization
Boc t-Butoxycarbonyl
Cmt chemically modified tetracycline
DIC N,N’-diisopropylcarbodiimide
DIPEA N,N-diisopropylamine
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide
EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide
ESI electron spray ionization
HATU O-(7-azabenzo-triazol-1-yl)-N,N,N ,N -tetramethyluronium hexafluorophosphate)
HMBC heteronuclear multiple bond correlation spectroscopy
HMQC heteronuclear multiple quantum correlation spectroscopy
HOAt 1-hydroxyazabenzo- triazole
HOBt 1-hydroxybenxtriazole hydrate
HR-EIMS high resolution electron impact mass
IR infrared spectroscopy
MHz megahertz
MIC minimal inhibitory concentration
MS mass spectrometry
NaCNBH3 Sodium cyanoborohydride
NMP N-Methyl-2-pyrrolidone
NMR Nuclear magnetic resonance
Pd (PPh3)4 tetrakis(triphenylphosphine)palladium (I)
Pd(OAc)2 palladium acetate
PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
TFA trifluoro acetic acid
THF tetrahydrofurane
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Curriculum Vitae
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Curriculum Vitae
Personal information
Surname, First name Usai Igor
Nationality Italian
Place / Date of Birth Cagliari, 3 August 1979
Gender Male
Education
10.2004 – 07.2008 Ph.D. Medicinal Chemistry
Advisor : Prof. Dr. Peter Gmeiner,
University Nürnberg-Erlangen (Germany) 1998 – 2004 University of Cagliari (Italy)
Pharmaceutical Chemistry & Technology
1993-1998 Secondary school : Liceo Scientifico “A. Pacinotti “, Cagliari
(Maturità Scientifica)
1985-1993 Primary and first three years of secondary school in Cagliari