Synthesis of Novel Bioactive Doxycycline Derivatives

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


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


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.


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.


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).


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


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.


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).


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).


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.


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.


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)


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)


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.


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)


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).


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.


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


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.


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.


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).


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).


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.


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).


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.


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.


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).


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).


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).


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).


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).


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).


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.


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.


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).


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.


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.


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.


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


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.


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.


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.


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.


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.


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.


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


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).


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).


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.


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.


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.


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.


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)


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.


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.


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


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.


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.


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.


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.


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.


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.


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


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.


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.


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.


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


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


3O

CONH2


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


n

O

NH2

ONH2

O

3

OHOOHO

HO

CH3OH

OHH2NOC

N

NH

O

R

n

OH

NH

O

nOOHO

HO

CH3OH

OHH2NOC

N


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


3O

CONH2


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) :


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


8.3 Hz, H-8), 7.48 (d, 1H, J = 8.3 Hz, H-7).


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) :




8.2 Hz, H-7), 7.91 (d, 1H, J = 8.2 Hz, H-8).


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) :


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).


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


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).


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) :


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).


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).


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).


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





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).


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).


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




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).


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).


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).


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).


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).


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).


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,


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


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).


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,


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





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).


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).


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)


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


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).


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)


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) :




7.9 Hz, H-7), 7.80 (d, 1H, J = 7.9 Hz, H-8).


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


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) :


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).


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)


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


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) :


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).


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) :




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





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’).


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, 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).


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)


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


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


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).


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)


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


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


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


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).


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)


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


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).


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)




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


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).


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)


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


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).


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)




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


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).


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)


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).


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)




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


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).


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


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


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)


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


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).


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


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).


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)


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


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).


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)



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


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



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.




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


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)



equivalents (0.068 mmol, 0.007 mL) of 5-hexynoic acid and 17 eq of DIPEA (0.578 mmol, 0.1 mL).





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


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)



equivalents (0.068 mmol, 10 mg) of N-Boc-propargylamine and 17 eq of DIPEA (0.578 mmol, 0.1 mL).





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


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).


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)



equivalents (0.068 mmol, 0.004 mL) of propargylamine and 17 eq of DIPEA (0.578 mmol, 0.1 mL).





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)


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).


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)


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)


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).


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)


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).


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)



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


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)



equivalents of 65 (MW = 214,22, 15 mg)) in 1 mL of dry DMF, and 17 eq of DIPEA (0.614 mmol, 0.1


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


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).


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)



equivalents of 66 (MW = 290,32, 21 mg) in 1 mL of dry DMF, and 17 eq of DIPEA (0.614 mmol, 0.1


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


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


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)



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


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


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)



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


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



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),



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)



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


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


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).


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


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)


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


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),


(S)-Methyl 2-[5-[4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-1-yl] pentanamido]-3-phenylpropanoate (75)


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).


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)


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) :


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


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).


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


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


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)


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


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).


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


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)



DMF. To this solution were added 2 equivalents of 79 (MW = 171.32, 12 mg) in 1 mL of dry DMF, and


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



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)



DMF. To this solution were added 2 equivalents of 80 (MW = 247.22, 18 mg) in 1 mL of dry DMF, and


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



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)



DMF. To this solution were added 2 equivalents of 81 (MW = 228,32, 16 mg) in 1 mL of dry DMF, and


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


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


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) :


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


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


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


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


SCH3), 1750-1600 (C=O), 1589 (C=C), 1528 (C=C) cm-1



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


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


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


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


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


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


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


SCH3), 1750-1600 (C=O), 1595 (C=C), 1537 (C=C) cm-1

Experimental Part

195

93


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


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


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

205

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

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