NEW CLASSES OF POTENTIAL MATRIX
METALLOPROTEINASE INHIBITORS BASED ON
OXAMATE, CARBAMOYLPHOSPHONATE AND
BISPHOSPHONATE FUNCTIONS
Dissertation
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
-Dr. rer. nat.-
dem Fachbereich 2 (Chemie/Biologie)
der Universität Bremen
vorgelegt von
Hanna Skarpos
2007
NEW CLASSES OF POTENTIAL MATRIX
METALLOPROTEINASE INHIBITORS BASED ON
OXAMATE, CARBAMOYLPHOSPHONATE AND
BISPHOSPHONATE FUNCTIONS
Thesis for
A Doctor’s degree
- Ph. D. -
Department of Chemistry
University of Bremen
of
Hanna Skarpos
2007
This thesis was carried out in Insitut of Inorganic and Physical Chemistry,
at University of Bremen from October 2004 till December 2007.
1st reviewer:
Prof. Dr. G.-V. Röschenthaler
2nd reviewer:
Prof. Dr. H. J. Breunig
Date of doctoral examination:
20. 12. 2007
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Table of contents
Table of contents
Chapter 1
Introduction .................................................................................13
Chapter 2
Aim of the study ..........................................................................20
Chapter 3
Results and discussion ...............................................................25
3.1
N-oxalyl and N-phosphonoformyl derivatives of Į-Tfm-Į-AAs- potential inhibitors of MMPs ...................................................25
3.1.1
Unnatural amino acids ..............................................................25
3.1.2
Oxamic and phosphonoformyl acids ........................................26
3.1.3
Fluorinated amino acids (FAAs) ...............................................29
3.1.3.1
Synthesis of methytrifluoropyruvate (MTFP) ............................31
3.1.4
Chemistry of new fluorinated MTFP imines containing
N-oxalyl and N- phosphonformyl groups ..................................32
3.1.4.1
Reaction of MTFP imines containing N-oxalyl and
N-phosphonformyl groups with Grignard reagents ...................35
3.1.4.2
Reaction of MTFP imines containing N-oxalyl and
N-phosphonformyl groups with ʌ-donors heteroaromatic
compounds................................................................................37
3.1.4.2.1 X-ray crystallographic analysis of methyl N-[ethoxyoxalyl]3,3,3-trifluoro-2-(1H- indol-3-yl)alaninate ..................................43
3.1.4.2.2 X-ray crystallographic analysis of methyl N-[ethoxyoxalyl]3,3,3-trifluoro-2-(2-furyl)alaninate .............................................45
-7-
Table of contents
3.1.4.3
Reduction reactions of MTFP imines possessing N-oxalyl and
N-phosphono formyl groups .....................................................47
3.1.4.4
Methyl 3-(diethylphosphonyl-2-(trifluoro-methyl-2H-azirine)formation and characteristic features .......................................48
3.1.4.5
Hydrolysis of the ester group in Į-Tfm-Į-amino acids bearing
a N-oxalyl group .......................................................................52
3.2
Nitrogen-containing bisphosphonates (N-BPs)-potential
inhibitors of MMPs ....................................................................53
3.2.1
Bisphosphonates (BPs)-general remarks .................................53
3.2.1.1
Chemical structure of BPs. Structure-activity relationships ......55
3.2.1.2
Nitrogen-containing bisphosphonates (N-BPs) - characteristic
features ....................................................................................56
3.2.2
Synthesis of nitrogen-containing bisphosphonates (N-BPs)
via click methodology ..............................................................59
3.2.2.1
Click chemistry - general remarks ............................................59
3.2.2.2
Synthesis of mono- and bispropargyl BPs ................................62
3.2.2.2.1 X-ray crystallographic analysis of tetraethyl hepta-1,6-diyne
-4,4-diyldiphosphonate .............................................................66
3.2.2.3
Synthesis of azides (R-N3) .......................................................67
3.2.2.4
An active copper(I) catalyst ......................................................70
3.2.2.5
1,3-Dipolar cycloaddition between monopropragyl
bisposphonate and different azides .........................................72
3.2.2.5.1 Catalytic Cycle for the Cu(I) catalyzed 1,3-cycloaddition ..........74
-8-
Table of contents
3.2.2.5.2 Triazoles - characteristic features ............................................78
3.2.2.6
Synthesis of nitrogen-bisphosphonates (N-BPs)
containing ditriazoles ................................................................79
3.2.2.6.1 X-ray crystallographic analysis of tetraethyl 2-(1-benzyl-1H1,2,3-triazol-4-yl)ethane-1,1-diyldiphoshonate .........................81
3.2.2.7
Click chemistry in situ ...............................................................82
3.2.2.8
Hydrolysis of the ester groups in N-BPs ...................................84
3.3
Fluorinated bisphosphonates (FBPs) - potential inhibitors of
MMPs ........................................................................................87
3.3.1
Fluorinated bisphosphonates (FBPs) - general remarks ..........87
3.3.2
A facile synthetic way to dilfuoro (phosphonooxy)vinyl
phosphonates ...........................................................................90
3.3.3
Studies concerning the reaction mechanism ...........................94
3.3.4
Hydrolysis of ester groups on phosphorus ...............................96
Chapter 4
Experimental section ................................................................98
4.1
General methods ......................................................................98
4.2
Physical methods .....................................................................99
4.3
Synthesis of compounds and their analytical data .................100
Chapter 5
X-ray data ...............................................................................140
Chapter 6
Summary ................................................................................170
-9-
Table of contents
Chapter 7
References .............................................................................176
Chapter 8
List of compounds ..................................................................187
Chapter 9
Appendix ................................................................................196
- 10 -
List of abbreviations
List of abbreviations
Å
angstrom
AZT
3’-azido-3’deoxythymidine
b.p.
boiling point
BP
bisphosphonate
BPs
bisphosphonates
i-Bu
isobutyl
t-BuOH
tert-Butanol
Bz
benzyl
CDCl3
deutero chloroform
CD3CN
deutero acetonitril
d
doublet
DFT
Density functional theory
DMF
dimethyformamide
DMSO
dimethysulfoxide
ECM
extracellular matrix
EtOAc
ethyl acetate
FAA
fluorinated amino acid
FAAs
fluorinated amino acids
g
gram
h
hour
HA
hydroxyapatite
HFPO
hexafluoropropene
m
multiplet
Me
methyl
- 11 -
List of abbreviations
mg
milligram
m.p.
melting point
MMP
matrix metalloproteinase
MMPs
matrix metalloproteinases
MMPIs
matrix metalloproteinases inhibitors
MTFP
methyltrifluoropyruvate
N-BPs
nitrogen containing bisphosphonates
Ph
phenyl
PPi
pyrophosphonates
ppm
parts per million
q
quartet
r.t.
room temperature
s
singlet
SARS
Structure-Activity Relationships
t
triplet
T
temperature
THF
tetrahydrofuran
ZBG
zinc binding group
- 12 -
Introduction
Chapter 1
Introduction
Nowadays, the ultimate goal of many chemists all around the world has
become rational or at least semi-rational drug discovery. Furthermore, the
development of the medicinal chemistry as both a pure and an applied science is
considered to have a significant impact upon it 1. A boost of medicinal chemistry is
based on development of such disciplines like combinatorial chemistry, compounds
identification (e.g. NMR), automated synthesis etc., but also organo-fluorine and
organo-phosphorus chemistry, which recently have taken an important effect on the
synthesis and design of a wide variety of biologically active compounds.
First of all, combinatorial chemistry has become recently one of the most
important methodologies in the field of medicinal and pharmaceutical chemistry.
It was developed by scientist, whose aim was to reduce costs and time connected
with designing new and effective drugs by taking into account the fact that finding a
novel drug is very often associated with a complex and complicated process.
Combinatorial chemistry bases on rapid synthesis and computer simulation. In
addition, it focuses on “one compound per well” instead of complex mixture of
compounds.
Secondly, the importance of fluorine in medicinal chemistry is well recognized.
Indeed, an increasing number of drugs on the market contain fluorine, the presence
of which often is of major importance to activity 2. The introduction of the fluorine
or perfluoroalkyl groups into molecules as well as the replacement of the hydrogen
atom and/or hydroxyl group by the fluorine atom is a widely used strategy in drug
development 3,4. The advantages in designing new pharmaceuticals are related to the
special properties of fluorine: its small size and high electronegativity. Fluorine
substitution in a drug molecule can influence not only physicochemical properties but
also pharmacokinetic properties like absorption, tissue distribution, secretion,
- 13 -
Introduction
pharmacodynamics and toxicology [Figure 1] 5. At present more than two hundred
pharmaceuticals containing fluor are available and new are appearing 6. They have
found application as volatile anaesthetics 7, as antifungal 8, antibacterial 9, antiinflammatory
10
, anti-depressant compounds
11
etc. Moreover, it was discovered that
fluorinated analogues of naturally occurring biologically compounds including amino
acids exhibit unique activities 12.
FLUORINE SUBSTITUTION
PHYSICOCHEMICAL
PROPERTIES
- bond strength
- lipophilicity
- conformation
- electrostatic
potential
- dipole and pKa
PHARMACOKINETIC
PROPERTIES
- tissue distribution
- clearance
- route of metabolism
- rate of metabolism
PHARMACOLOGICAL
CONSEQUENCES
- pharmacodynamics
- toxicology
Figure 1 The effect of fluorine substitution on drug discovery
Thirdly, organophosphorus chemistry, being ignored for many years,
recently has achieved important and well-recognized place in the search for new
drugs
13
. Organophosphorus chemistry, as a discrete area of study, is the study of
compounds containing a C-P bond
14
. Its present impact on the field of medicinal
chemistry is even difficult to quantify. Among the list of all organophosphorus
compounds the main place is occupied by phosphonates and bisphosphonates,
which have found huge application as pharmaceuticals. For instance, derivatives of
- 14 -
Introduction
phosphonic acid are used in the synthesis of different alpha-aminophosphonic acids
which are considered to be structural analogues of the corrensponding alpha-amino
acids. It is noteworthy that, their negligible mammalian toxicity, and the fact that they
very efficiently mimic aminocarboxylic acids makes them extremely important
antimetabolites in the process of designing new drugs
15
. On the other side,
bisphosphonates have been found recently as an ideal therapeutic agent for
treatment of different bone diseases. Their capability to chelate metal ions and inhibit
crystals growth but also a strong affinity to bone was used in synthesizing new drugs
by many pharmaceuticals companies 16.
Interestingly, the list of compounds designed and synthesized as new drugs,
every year is quite long and various. Without any doubt, highlights of this list are
matrix metalloproteinase inhibitors (MMPIs). Recent evidences concerning MMPIs as
potential drugs in the treatment of different pathologies have expanded considerable
attention.
There
is
a
presumption
that
the
main
focus
of
research
in nearest futures will belong to the development of novel, less toxic and more
effective inhibitors of MMPs. Mainly, they seem to be attractive cancer target; they
have multiple signaling activities that are commonly altered during tumorgenesis that
might serve as intervention points for anticancer drugs
17
. Moreover, MMPs are
involved in the regulation of bone remodeling. It is commonly known that the activity
of these enzymes is important for activating osteoclasts to resorb bone
18
. Thus, they
can represent a potential class of drugs for pharmacological treatment of bone
diseases, like osteoporosis. Remarkable, MMPs have been used to cure
cardiovascular diseases
19
. Despite of this, they are useful targets in treatment of
arthritis, restenosis, pulmonary emphysema, multiple sclerosis and stroke 20.
Matrix metalloproteinases (MMPs), also called matrixins, are a class of Zncontaining , Ca2+- dependent multifunctional proteins with major functions in the
degredation and remodeling of all components of the extracellular matrix (*),
21-23
.
MMPs are regulated by hormones, growth factors, and cytokines, and are involved
in ovarian functions. Endogenous MMP inhibitors (MMPIs) and tissue inhibitors
of MMPs (TIMPs) strictly control these enzymes. Overexpression of MMPs results
in an imbalance between the activity of MMPs and TIMPs that can lead to a variety
of pathological disorders like: Alzheimer’s disease, cancer, arthritis, rheumatoid
etc 25,26.
- 15 -
Introduction
Currently, 29 human matrix metalloproteinases has been identified. MMPs can
be classified according to their substrates into: collagenases, gelatinases,
stromelysins, membrane type MMPs (MT-MMPs) and matrilysin
(**), 27
[Figure 2].
As mentioned before, MMPs contain Zn atom which structural role is essential for
understanding the mechanism of action of these proteinases. The Zn2+ ion present
at the active sites of these enzymes is crucial for enzymatic activity. Therefore,
virtually all attempts to develop inhibitors have been based on “ion-binding-groups”
(ZBG) 28.
Figure 2 Structural domains of MMPs
Initially, the amino acid sequence around the collagenase cleavage in collagen
has been taken as the guide for the substrate-based design approach for synthetic
inhibitors
23
. However, many of the unnatural amino acids - inhibitors of the MMP
were at the same time designed and optimized 29,30.
------------------------------------* The extracellular matrix (ECM) is a complex structural entity surrounding organs and tissues of the
human body. The ECM plays role in cell-cell signalling, wound repair, and cell adhesion and tissues
24
formation .
** See Chapter 9- Appendix, List of MMPs
- 16 -
Introduction
Later, it was predicted that the requirement for a molecule to be an effective inhibitor
of this class of enzymes is a functional group (ZBG). Furthermore, at least one
functional group should provide a hydrogen bond interaction with the enzyme
backbone, and one or more side chains should undergo effective van der Waals
interaction with the enzymesubsites
28
. By using this approach, many scientist
groups’ taking advantage from combinatorial chemistry and structure- based design,
have started to discover a number of different ZBG functional groups. Hitherto, many
new potent inhibitors of MMPs have been synthesized and their excellent examples
are derivatives of hydroxamic, carboxylic, phosphonic, carbamoylphosphonic and
bisphosphonic acids etc.
Among this list, the overwhelming majority belongs to a hydroxamic acid. Its
low toxicity and ability to form abidentate chelate with the zinc atom in the enzyme’s
active site is considered to be an important functional feature metalloenzyme
inhibition, namely as inhibitors of metalloproteinase
31
. Over the last decades
compounds containing derivatives of hydroxamic acid have undergone rapid clinical
development. As a result of many studies, on the market appeared new anticancer
drugs
32
(inhibitors of MMPs): Batimastat (BB-94)
12-9566
35
33
, Marimastat (BB-2516)
34
, Bay
[Figure 3].
S
S
H
O
O
H
H
H
N
HO
N
O
H
H
CH 3
N
H
OH
O
O
M arimastat
Barimastat
S
O
OH
O
Cl
Bay 12-9566
Figure 3 Hydroxamate Inbibitors of MMPs: Barimastat, Marimastat and Bay 12-9566
- 17 -
O
N
N
N
HO
H
H
Introduction
Inhibitors based on phosphorus ZBGs have been for a long time a favoured area
of investigation
38
. Recently, Breuer et al.
39,40
have introduced a new class of in vivo
active MMPIs: alkyl- and cycloalkylcarbamoyl phosphonic acids which possess
significant antiangiogenic activity, which enhances the anticancer effect of the drugs.
Examined by them the acylphosphonic acid (oxophosphonic acids) derivatives have
shown to inhibit MMP-2. Noteworthy, the acylphosphonic group is capable to chelate
calcium with the formation of the five-membered ring, resembling the mode of zinc
chelation by a hydroxamate function in MMP inhibitors 41.
In addition, it was discovered that various matrix metalloproteinases are
inhibited in vitro by several bisphosphonates
41
. Recently, Nakaya and co-workers
42
have demonstrated an inhibitory effect of bisphosphonates (especially Tiludronate)
on the activity of both MMP-1 and MMP-3. Going into details, bisphosphonates have
shown to inhibit host degradative enzymes in human periodontal ligament cells.
Their results support the continued investigation of these drugs as potential
therapeutic agents in treatment of periodontal diseases. Additional, bisphosphonates
have been found to inhibit tumour growth and metastasis in some tumours such
as breast cancer. Cheng et al.
43
demonstrated that Alendronate downregulates
MMP-2 secretion and induces apoptosis in osteosarcoma cells, which may both
contribute to the reduction of invasive potential of the tumour cells [Figure 4].
O
O
OH
HO
P
H 2N
OH
HO
OH
OH
P
Cl
S
OH
O
P
OH
OH
P
OH
O
Figure 4 Bisphosphonate inhibitors of MMPs: Alendronate and Tiludronate
According to many scientists bisphosphonates possessing low toxicity and being
tolerated by humans have the potential to become one of the most popular matrix
- 18 -
Introduction
metalloproteinase inhibitors for MMP-related human soft and hard tissue destructive
diseases in nearest future 43.
In summary, currently there is an acute need for the design of new ZBGs
which can become a new generation of drugs useful on the life-long treatment of
many diseases characterized by excessive remodelling of degradation of ECM such
as tumor, rheumatoid arthritis, osteoporosis etc. Furthermore, there is a need for new
strategies which can be employed to design effective drugs such as inhibitors of
matrix metalloproteinase and at the same time can highlight the strenghts and
drawbacks of each approach. Obviously, the design of the MMP inhibitors
demonstrates the synergy between medicinal chemistry, structural biology and
molecular remodelling. Using this approach, the development of effective matrix
metalloproteinase inhibitors can consider the main focus of research efforts in the
future.
- 19 -
Aim of the study
Chapter 2
Aim of the study
In recent years, many efforts have been spent on the development
of the inhibitors of matrix metalloproteinase. Experimental evidence confirms that
they play a significant role in a treatment of serious pathologies. For instance, nearly
three- decade long trials for discovering MMP inhibitors with anticancer efficiency
have brought only disappointing results. Nowadays, many groups dissect different
classes of zinc chelators (ZBG). Till now, several peptide and non-peptide based
ZBGs have been identified like: hydroxymate, carboxylate, derivatives of phosphoric
acid, derivatives of sulphuric acid and thiols [Figure 5]. Obviously, the new
challenges include the development of new inhibitors bearing more effective ZBGs as
well as the development of new allosteric non-zinc binding inhibitors 44.
O
O
O
HO
HO
P
N
HO
O
HO
P
HO
H
O
O
HN
HO
P
NH
O
S
S
SH
N
H
N
H
Figure 5 ZBGs (Zinc- Binding groups) commonly used in designing of MMPIs
The specific aim of this thesis was to design and synthesize a new generation
of matrix metalloproteinase inhibitors which could be used for the treatment of many
pathologies. In another words, we were interested in synthesizing potent drug
- 20 -
Aim of the study
candidates and characterizing them from chemical point of view. Indeed, we paid our
attention on finding facile routes for the synthesis of compounds which can possess
properties of inhibiting the action of secreted enzymes involved in connective tissue
breakdown such as collagenases, gelatinases, and stromelysin etc. Furthermore,
it has been our an ongoing aim to develop highly efficient methods for introducing
to the new ZBGs fluorine atoms or fluorinated groups by taking into account that their
presence in the molecule is bringing excellent changes. In particular, in terms of drug
design, fluorine substitution can be used to alter the rate of drug metabolism and
thereby produce a drug with a longer duration of action 5.
The first part (Chapter 3.1) focuses on syntheses of the compounds
possessing N-oxalyl and N-phosphonoformyl groups- potential candidate molecules
for the design of novel inhibitors of medically important enzymes. Our library design
of these molecules firstly we planned by the synthesis of new imines of methyl
trifluoropyruvates bearing ZBGs at the nitrogen [Scheme 1, entries 7, 8, Route A],
based on the reaction of methyltrifluoropyruvate (2) with ethyloxamate (3) and diethyl
carbamoylphosphonate (4), respectively, followed by dehydratation. Furthermore,
reaction of imines with different nucleophiles [Scheme 1, Route B] could easily
transform them into useful synthetic intermediates (fluorinated amino acid
derivatives-potential drug derivatives), taking into account their electrophilic
character.
O
O
O
O
R
F3C
OEt
+
OMe
O
H2N
A
OEt
3
F3C
O
OEt
OMe
O
+ H2N
A
O
OEt
OEt
N
P
P
OEt
MeO2C
O
O
2
O
R
F3C
F3C
N
H
CO2Me
O
7
O
O
OEt
B
N
MeO2C
O
2
F3C
B
OEt
F3C
N
H
CO2Me
P
OEt
O
8
4
Scheme 1 Design of N-oxalyl and N-phosphonoformyl derivatives of Į-Tfm-Į-amino acids - potential
inhibitors of MMPs
- 21 -
Aim of the study
In addition, our outgoing aim was to develop highly efficient strategies for the
synthesis of unnatural amino acids possessing in the position Į-trifluoromethyl group.
Interestingly, Į-amino acids containing Tfm groups are of particular interest due to its
unique characteristic, such as high electronegativity, electron density etc. Obviously it
can serve as a “final push” towards higher activity and stability after rational design.
The second part (Chapter 3.2) concerns the chemistry of bisphosphonates.
Recently, they have become promising targets in inhibiting MMPs, for example
Alendronate is capable to inhibit MMP-2
43
. We strongly believe that synthesis of the
new derivatives of bisphosphonates could bring novel family of highly potent drugs
useful in the treatment of much pathology. Among all the commonly known methods,
the approach based on the Cu-catalyzed azide-alkyne 1,3-dipolar cycloaddition
(CuAAC) introduced recently by Sharpless
135,136
seems to be highly versatile and
effective choices for rapid synthesis. Moreover, we assumed that click chemistry
could give us high-throughput strategy for assembling bisphosphonate molecules
with different compounds. It is noteworthy to add that this methodology is providing
a subtle method for introducing triazoles to the molecules of bisphosphonates, which
can be mimicked by peptides. Our library design of functionalized bisphosphonate we
planned to base by the synthesis of mono- and diproparygl bisphosphonates- first
counterparts for click reaction and different azides- second counterpart [Figure 6].
O
O
OEt
OEt
P
P
P
OEt
OEt
P
OEt
R-N3
OEt
OEt
O
O
Monopropargyl bisphosphonate
OEt
Bispropargyl bisphosphonate
Azides
Figure 6 Counterparts for Cu-catalyzed azide-alkyne 1,3-dipolar cycloaddition
Next, by using different method of introducing Cu(I)-active catalysts into 1,3-dipolar
cycloaddition between above presented counterparts we could find an general
- 22 -
Aim of the study
synthetic approach giving the possibility for facile and rapid synthesis of different Nbisphosphonates-potential inhibitors of MMPs [Scheme 2].
O
O
OEt
OEt
P
P
P
OEt
OEt
R-N3 , Cu(I)
OEt
N
R
OEt
OEt
P
N
OEt
N
O
O
37
Scheme 2 A general synthetic approach to R-functionalized nitrogen containing bisphosphonates
(N-BPs)
Continously, the reaction between bispropargyl bisphosphonate and different azides
could
allow us
to
introduce
different functionalities
[Scheme
3,
R]
into
bisphosphonate molecule through a triazole heterocycle.
O
EtO
OEt
P
P
OEt
EtO
O
O
P
P
OEt
OEt
R-N3 , Cu(I)
OEt
OEt
N
O
R
N
N
N
N
N
R
38
Scheme 3 A general synthetic approach to R-functionalized nitrogen-bisphosphonates (NBPs)
containing two triazoles moieties
Finally, we planned also to focus on the one-pot reaction, in which azides are
synthesized in situ [Scheme 4].
- 23 -
Aim of the study
O
O
OEt
OEt
P
P
OEt
OEt
P
NaN3 , RBr
OEt
, Cu(I)
one pot reaction
N
R
OEt
P
N
OEt
OEt
N
O
O
37
Scheme 4 A general synthetic approach to nitrogen-bisphosphonates (N-BPs) based on one pot
reaction
Besides, the aim of our study was design and synthesis of the new class of
fluorinated bisphosphonates due to the escellent potential of fluorine (Chapter 3.3).
In order to design novel class of bisphosphonates containing fluorine atoms or
fluorine groups we planned to base on the Michael-Arbuzov/ Perkov reaction
[Scheme 5].
O
F
P(OR)3
+
O
P
C
ClCF2COCl
F
P
OR
OR
OR
OR
O
(R= SiMe3 , Et)
Scheme 5 A general synthetic approach to fluorinated bisphosphonates (FBPs) based on Arbuzov/
Perkov reaction
- 24 -
Results and discussion
Chapter 3
Results and discussion
This chapter focuses on the presentation of our achievements in designing
new potential inhibitors of matrix metalloproteinases possessing interesting ZBGs.
Each sub-chapter contains general remarks concerning a given class of compounds
and the discussion connected with the presented results. The chapter is divided into
three parts due to the different ZBGs: synthesis of N-oxalyl and N-phosphonoformyl
derivatives of Į-Tfm Į-amino acids, synthesis of a new class of nitrogen containing
bisphosphonates and finally synthesis of a new fluorinated bisphosphonates.
3.1 N-Oxalyl and N-phosphonoformyl derivatives of Į-Tfm Į-amino
acids - potential inhibitors of MMPs
3.1.1 Unnatural amino acids
The interest in amino acids (AAs) and their derivatives has existed for many
years
45
. Amino acids are the basic building units (monomers) of proteins. They
possess ability to form shorter or longer polymers called peptides or polypeptides.
A molecule of amino acid contains an amino (-NH2) and carboxyl groups (-COOH)
attached to the same Į-carbon (this term refers to alpha-amino acids) - [Figure 7].
Besides, amino acids differ from each other the side chain (R).
- 25 -
Results and discussion
Figure 7 Structure of amino acids (AAs)
The last decade has witnessed a great revolution of chemists and biochemists giving
the ability to incorporate the unnatural amino acids into proteins (different from the
canonical twenty). That has become a powerful tool for illuminating the principles
of protein design. Moreover, peptides modified by non-proteinogenic amino acids are
useful building blocks for drug discovery
that
unnatural
amino
metalloproteinases
acids
46
. There are many evidences suggesting
possess
high
affinity
in
inhibiting
matrix
47-50
. Therefore, the development of new synthetic pathways to
unnatural amino acids containing different functionalities remains a constant
challenge 51.
3.1.2 Oxamic and phosphonoformic acid
Because of our major interest in designing compounds with functional groups
capable of binding to the zinc(II) site in the MMPs we decided to examine two
amines: ethyl oxamate and diethylcarbamoylphosphonate [Figure 8].
- 26 -
Results and discussion
O
O
OEt
OEt
H2N
H2N
P
OEt
O
O
Diethyl carbamoylphosphonate
Ethyl oxamate
Figure 8 Ethyl oxamate and diethylcarbamoylphosphonate - active ZBGs
In general, both oxamic and phosphonoformic acid possess interesting ligating
possibilities which play a crucial role in inhibition of matrix metalloproteinases. In this
connection, hydroxamic acid derivatives like oxaloglycine are commonly known to
inhibit prolyl hydroxylase involved in the biosynthesis of collagen
52,53
. On the other
side, phosphonoformic acid derivatives like phosphonoformate (Foscarnet) are
effective antiviral compounds medicinally used in the treatment of AIDS (acquired
immune deficiency syndrome), HSV-1 and HSV-2 (herpes simplex virus), CMV
(cytomegalosvirus) retinitis etc
54,55
. It is noteworthy to add, that acylphosphonates
are capable to interfere with biological processes involving calcium and
hydroxyapatite
56
. The acylphosphonic group (1-oxophosphonic), in particular
carbamoylphosphonic, was shown to chelate calcium with the formation of a fivemembered ring [Figure 9], resembling the mode of zinc chelation by a hydroxamate
function in MMP inhibitors
57-59
. Interestingly, carbamoylphosphonates are potent
inhibitors of MMP, while phosphonic acid lacking the Į-carbonyl groups does not
inhibit MMPs.
- 27 -
Results and discussion
O
O
R
H
N
C
P
O
O
R
O
H
N
M
C
O
P
O
O
O
R
H
N
C
P
O
O
O
M
M
I
III
II
M = Ca, Mg, Zn, Cu
Figure 9 Coordination of the carbamoylphosphonate to a metal ion: (I) monodentate manner, (II) fourmembered ring, (III) five-membered ring between the oxygen’s of the two functional groups
Ethyl oxamate for our experiments was purchased from Sigma- Aldrich company,
while diethylcarbamoylphosphonate was synthesized according to procedure given
by Breuer et al 39, 40 [Scheme 6].
O
O
Cl
1. P(OEt)3
2. NH3
OEt
THF
OEt
H2N
P
OEt
O
4
Scheme 6 Synthesis of an active ZBG-diethyl carbamoylphosphonate 4 via Arbuzov reaction
Reaction between one equivalent of ethyl chloroformate and one equivalent of triethyl
phosphite yields the phosphonate derivative. This reaction is referred as MichaelsArbuzov rearrangement. During this transformation tervalent phosphorus is
converted into pentavalent phosphorus. Next, the addition of the ammonia leads to
the desired carbamoylphosphonate, which can be used as a starting compound for
further transformations. The strong nucleophilic amino group is opening up the
possibility of this phosphonate to react with many different electrophilies like e.g.
methyltrifluoropyruvate (MTFP).
- 28 -
Results and discussion
3.1.3 Fluorinated amino acids (FAAs)
The man-made area of fluorinated amino acids (FAAs) takes the most
important place in the family of unusual aminoacids
45
. Most importantly, fluorinated
amino acids have recently emerged as valuable building blocks for designing
hyperstable protein folds, as well as directing highly specific protein-protein
interaction
46,60
. The synthesis and design of new fluorinated inhibitors of MMPs
containing side chains with fluorine atom or fluorine groups
61
has become an
important part in the field of biologically active organofluorine chemistry and is of
current interest. The side chain RF was found to be critical not only for potency but
could also dramatically influence the enzyme selectivity profile of the inhibition 62.
Interestingly, the introduction of fluorine atom or fluorine containing groups into
the desired molecule is very often accomplished with remarkable changes of its
biological and chemical activity. Obviously, the fluorine atom is not a sterically
demanding substituent (van der Waals radii r(F) = 1.35 Å, r(H) = 1.20 Å) and the C-F
bond is 0.4 Å longer that the C-H bond 63.
Moreover,
amino
acids
containing
trifluoromethyl
(Tfm)
groups
are
an attractive choice because they are reasonably isosteric to their nonfluorinated
counterparts
issue
64
. The steric bulk of a trifluoromethyl group is still a controversial
, however its presence in Į-position of an aminoacid exerts considerable
65
polarization effects on the neighbouring substituents
66
. This structural alternation
influences the hydrolytic stability of peptides containing Tfm amino acids resulting in
retarded degradation by peptidases and, consequently, in prolonged intrinsic activity.
In spite of this, due to the high electron density, the Tfm group is capable of
participating in hydrogen bonding as a hydrogen receptor
66-68
. This can provide
additional interactions within the MMPs active sites. Noteworthy, Tfm group can
inhibit potency of MMPs
69,70
. Furthermore, the large size and volume of Tfm groups,
in combination with the low polarizability of fluorine atoms leads to enhanced
hydrophobicity
71
. Finally, the incorporation of the fluorine into the molecule of AAs
brings opportunity to monitor their conformational properties and metabolic
processes.
- 29 -
Results and discussion
All fluorinated amino acids according to Qing et al.72 can be grouped into three main
classes [Figure 10]:
¾ Fluorinated Į-amino acids
ƒ
Monofluorinated Į-amino acids
ƒ
Gem-Difluoromethylated Į-amino acids
ƒ
Trifluoromethylated and polyfluorinated Į-amino acids
¾ Fluorinated ȕ-amino acids
ƒ
Monofluorinated ȕ-amino acids
ƒ
Gem-Difluoromethylated ȕ-amino acids
¾ Fluorinated cyclic amino acids
F3C
H2N
CF3
R
CO2R
F3C
CO2H
BzHN
N
CO2R
OH
Boc
R = Bz, Et, Me, Allyl 73
A
R = Me74
R = Me75
B
C
Figure 10 Main classes of Tfm-AAs; A: Trifluoromethylated Į-amino acids, B: Trifluoromethylated ȕ-amino
acids, C: Trifluoromethylated cyclic amino acids
Without any doubt, the class of trifluoromethylated Į-amino acids has been
considered as one of the most interesting biologically compounds taking into
consideration the fact that they can inhibit the action of various phosphate-dependent
enzymes, such as alanine racemases or decarboxylases 76-78.
Our efforts connected with designing unnatural amino acids possessing
abilities to inhibit MMPs concerned attachment of fluorinated counterpart, namely
trifluoromethylated group (Tfm) to oxamic and phosphonoformic moieties.
Most importantly, electrophilic methyl trifluoropyruvate imines are excellent
precursors for synthesis of different cyclic and acyclic Į-Tfm amino acids
- 30 -
79
.
Results and discussion
Moreover, the chemistry of fluorine- containing imines may rightfully be considered
as one of the most interesting areas of fluorine chemistry 80.
3.1.3.1 Synthesis of methytrifluoropyruvate (MTFP)
Recently, different methyl trifluoropyruvate imines, bearing on the nitrogen
atom different functional groups, have been synthesized [Figure 11] 81-84 from methyl
3,3,3-trifluoropyruvate (MTFP) and corresponding amines.
F3C
N
SO2R
OEt
N
H3COC
O
O
F3C
P
H3COC
OEt
O
R = Ph, Me
Figure 11 Methyl trifluoropyruvate imines possessing sulfonyl and phosphoryl groups
Methyl 3,3,3-trifluoropyruvate is the methyl ester of trifluoropyruvic acid. Due to
the reactive nature of the alpha carbonyl group it has been extensively applied as
a fluorinating block in synthesis of heterocyclic derivatives and unnatural amino
acids. The preparation of methyl 3,3,3-trifluoropyruvate in our laboratory was
accomplished according to the standard procedures 85-87 [Scheme 7].
- 31 -
Results and discussion
O
F3C
F
F
O
F3C
CH3OH
OCH3
H2SO4
F3C
OCH3
F
O
F
A
1
OCH3
O
B
1a
2
Scheme 7 Synthesis of methyl 2,3,3,3-tetrafluoro-2-methoxypropanoate by methanolysis of
1,1,2,3,3,3- hexafluoropropanoate (HFPO) (A); Synthesis of methyl 3,3,3trifluorpyruvate from methyl 2,3,3,3-tetrafluoro-2-methoxypropanoate (B)
To be more precise, this synthesis consists of two steps. At the beginning,
commercially
available
hexafluoroprepene-1,2-oxide
(HFPO) (1)
reacts
with
methanol giving the product of the methanolysis - methyl 2,3,3,3-tetrafluoro-2methoxypropanoate (1a). Then the addition of concentrated sulphuric acid (96%)
leads to the desired methyl 3,3,3-trifluoropyruvate (2).
3.1.4
Chemistry
of
new
fluorinated
imines
containing
N-oxalyl
and
phosphonoformyl groups
In our studies, we found that methyltrifluoropyruvate (2) had reacted readily
with amines: ethyl oxamate (3) and diethylcarbamoylphosphonate (4) [Scheme 8, 9]
at room temperature in the absence of any solvent giving analytically pure amines (5)
and (6) in a high yield [Scheme 8, 9]. Both amines are stable in a dry inert
atmosphere. Next, we investigated the conversion of the amines into imines.
According to some data
80,88
, the dehydratation conditions for hemiaminal
compounds are directly dependent on the electron-accepting properties of the
substituents at the ketone carbonyl carbon atom and the basicity of the amino group.
Taking into consideration above mentioned, we followed the procedure proposed by
Osipov and Burger
89,90
using trifluoroacetic anhydride/pyridine as dehydrating
agent. As a result of the rapid transformation of the both hemiamidals of
- 32 -
Results and discussion
methyltrifluoropyruvates, we afforded the corresponding imines (7) and (8) [Scheme
8, 9]. Obtained imines were purified by distillation.
O
F3C
F3C
CO2Me
OEt
OEt
r.t
H2N
MeO2C
N
H
no solvent
O
O
OH
O
2
O
5
3
CF3
(98%)
O
OEt
TFAA/Py
MeO2C
N
Et2O, 0oC
O
7
(97%)
Scheme 8 Synthetic route to methyltrilfuoropyruvate imine 7 bearing on nitrogen potential ZBG,
namely oxalyl group
O
F3 C
F3C
CO2Me
OEt
H2N
P
O
O
OH
OEt
r.t
MeO2C
OEt
no solvent
N
H
P
OEt
O
2
O
6
4
CF3
(82%)
O
OEt
TFAA/Py
MeO2C
N
P
Et2O, 0oC
OEt
O
8
(72%)
Scheme 9 Synthetic route to methyltrilfuoropyruvate imine 8 bearing on nitrogen potential ZBG,
namely phosphonoformyl group
Imine (7) is a yellow/orange liguid- stable in dry atmosphere. The signal of the
19
F
NMR was observed in the range typical for such a compounds [δF: -71.3 (s, CF3)].
- 33 -
Results and discussion
However, in the case of the imine (8), we observed partly decomposition of the
product (§ 20%). Much of our effords for achieving completely pure product failed.
Indeed, imine (8) characterized by
F NMR [δ: -71.3 (s, CF3)] and
19
31
P NMR [δ: -2.2
(m)] was used as a crude product for further transformation.
Generally, imines have a planar trigonal framework of a sp2 carbon and a sp2
nitrogen atom. Each uses one sp2 orbital to form a ı bond to the other atom and a p
orbital to form a ʌ bond to the other atom. The carbon uses two sp2 orbitals and the
nitrogen one sp2 orbitalt to form ı bonds to the substituents (in our case three
powerful electron-withdrawing groups) [Figure 12].
p orbitals overlap to form a C-N "pi" bond
C-C "sigma" bond between sp2 orbitals on carbon and nitrogen
lone pair in
sp2 orbital
F3C
trigonal planar
sp2 carbon
C
trigonal planar
sp2 nitrogen
N
MeO2C
R
R = C(O)OEt; C(O)P(O)(OEt)2
Figure 12 Orbitals structures of methyltrilfuoropyruvate imines 7 and 8
As already mentioned, the new imines are stable only in inert atmosphere of nitrogen
because of their ability to become hydrolysed by water. The low stability of the new
imines can be explained by the presence of electropositive groups (acceptors) on the
nitrogen namely oxalyl and phosphonoformyl derivatives in contrast to stable oximes,
hydrazones and semicarbazones in which the nitrogen atom carries electronegative
groups. For instance oximes are more stable than typical imines because the
electronegative substituent can participate in delocalization of the imine double bond.
Delocalization decreases the į+ charge on the carbon atom of the imine double bond
C=N and raises the energy of the LUMO, making it less susceptible to nucleophilic
attack 91.
- 34 -
Results and discussion
Our aim was to study the characteristic features of the behaviour of the methyl
trifluoropyruvates imines possessing three powerful electron withdrawing groups and
synthesize their derivatives. To investigate our presumption concerning electrophilic
nature of methyl trifluoropyruvates imines we used the following reactions: reactions
with organometallic reagents, reactions with ʌ-donor heteroaromatic compounds and
reduction reactions.
3.1.4.1 Reactions of the methyltrifluoropyruvates (MTFP) imines containing Noxalyl and N-phosphonoformyl groups with Grignard reagents
Examining the nature of the new imines, firstly we focused on reaction
of imines with Grignard reagents; the addition of organometallics to imines is
a common approach to synthesize amines. There have been published numerous
literatures concerning the addition of the organometallic reagent to imines
92
and it is
commonly known that this addition can proceed normally on the carbon atom
(carbophilic addition). Interestingly, the addition of the organometallic reagent on the
nitrogen atom (azophilic addition) has been also described recently
experiment we observed, in analogy to other acylimines
89
93
. In our
of MTFP [Scheme 10] that
imine (7) and (8) reacted smoothly with different organometallic compounds of Mg
at -78oC leading to the formation of the new C-C bond.
- 35 -
Results and discussion
CF3
O
OEt
MeO2C
N
O
F3C
1. RMgCl
Et2O/ THF
-780C
7
2. H+, H2O
O
O
OEt
MeO2C
CF3
R
N
H
X
O
9- 16
OEt
MeO2C
P
N
X= C or P-OEt
OEt
R= Me, Ph, Bz, Allyl
O
8
Scheme 10 Reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines 7 and 8 with
different Grignard reagents
The nucleophilic addition of organometallic compounds to C=N double bond (NHydro-C-alkyl-addition) proceeded regiospecifically and resulted in the alkylation
of the Į-carbon, to give the desire derivatives of Į-Tfm-Į-amino (8-16) acids
in moderate to good yields.
The signal of the
19
F NMR was observed in the range typical for such compounds
[§ δF: -72.2 (s, CF3)], while the signal of the
P NMR was found arround [§ δP: -1.3
31
(m, P(O)(OEt)2)]. It should be noted that the products were purified by flash column
chromatography on silica gel using mixture of petroleum ether and ethyl acetate
as an eluent. These results are summarized in the table below [Table 1].
a
Entry
Product
R
X
Yield , %
1
8
Me
C
65
2
9
Me
P-OEt
58
3
11
Ph
C
52
12
Ph
P-OEt
49
4
- 36 -
Results and discussion
5
13
CH2Ph
C
59
6
14
CH2Ph
P-OEt
55
7
15
All
C
43
8
16
All
P-OEt
48
Table 1
Results of the reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines
with Grignard reagents via Scheme 10
a
yield are given after purification by column chromatography
3.1.4.2 Reactions of methyl trifluoropyruvates (MTFP) imines containing
N-oxalyl and N-phosphonoformyl groups with ʌ-donor heteroaromatic
compounds
Our next attempt in examining the nature of the imines (7) and (8) were
reactions with different ʌ-donor heteroaromatic compounds. In general, we
investigated the chemistry of the new imines as electrophiles. We presumed that they
can be sufficiently alkylated by different ʌ-donor heteroaromatic compounds.
Our first afford was the reaction with indoles. These benzo-fused pyrroles are
a very important class of heterocyclic systems, because they are built into proteins in
the form of the amino acid tryptophan. We found out that the reaction of imines (7)
and (8) with one- substituted indoles in anhydrous diethyl ether proceeded in mild
condition [Table 2, entries 17, 19] or at moderate heating [Table 2, entries 18, 20]
giving corresponding Į-aryl (hetaryl)-ȕ,ȕ,ȕ-trifluoroalanine [Scheme 11] preferably
substitued 3-position, as a result of the fact, that this reaction involves a rather
isolated enamine system in the five-membered ring and does not disturb the
aromaticity of the benzene ring.
- 37 -
Results and discussion
CF3
O
OEt
N
MeO2C
MeO2C
CF3
O
O
O
7
N
R
N
H
H
O
Et2O, r.t
CF3
N
O
R
H
OEt
N
MeO2C
X
P
17-20
OEt
R= H, CH3
X= C or P-OEt
O
8
Scheme 11 Reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines with indoles
The signal of the
19
F NMR was observed in the range typical for such compounds
[§ δF: -71.6 (s, CF3)] and consequently, the signal of the
31
P NMR spectra was
observed in the range typical for such compounds [§ δP: -1.2 (m, P(O)(OEt)2)]. These
results are summarized in the table below [Table 2].
Reaction
Temperature,
o
C
Yield,
%
Entry
Product
R
X
Y
1
17
H
C
-
r.t.
93
2
18
H
P-OEt
-
r.t.
85
3
19
Me
C
-
60
62
4
20
Me
P-OEt
-
60
50
Table 2
Results of reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines with
indols via Scheme 11
a
yield are given after purification by column chromatography
Similar to indoles, furan and N-methylpyrrole proceeded easily electrophilic
substitution by imines (7) and (8) [Scheme 12]. These reactions were carried out in
anhydrous diethylether at 0oC (then slowly warming up to room temperature) to
- 38 -
Results and discussion
receive the desired products - Į-aryl (hetaryl)-ȕ,ȕ,ȕ-trifluoroalanine in moderate yield
[Table 3, entries, 21-24].
CF3
O
OEt
MeO2C
N
O
O
CF3
7
O
Y
CF3
Et2O, O
O
RT
N
H
X
Y
CO2Me
O
OEt
MeO2C
21-24
P
N
OEt
X= C or P-OEt
O
R=O, N-Me
8
Scheme 12
Reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines with furan
and N-methylpyrrole
This 1-hetero-2, 4-cyclopentadienes containing a butadiene unit bridged by a sp2hybridized heteroatom undergo electrophilic substitution in two possible positions C2
and C3. Both modes benefit from the presence of the resonance-contributing atom,
however attack at the C2 is leading to an intermediate with additional resonance
form.
Indeed, the position C2 seemed to be more preferable site of electrophilic
substitution. We can point out that we have noticed such selectivity. By examining
the reaction the mixture we observed the main product belonging to the electrophilic
substitution on C2 and small amount of products belonging to electrophilic
substitution on C3.
The signal of the
19
F NMR was observed in the range typical for such compounds
[§ δF: -72.2 (s, CF3)] and consequently, the signal of the
31
P NMR was observed in
the range [§ δP: -1.5 (m, P(O)(OEt)2]. The reaction mixture was purified on silica gel
by flash column chromatography. The results are summarized in the table below
[Table 3].
- 39 -
Results and discussion
Reaction
Temperature,
o
C
Yield,
%
Entry
Product
R
X
Y
1
21
-
C
O
0ĺr.t.
39
2
22
-
P-OEt
O
0ĺr.t.
40
3
23
-
C
N-Me
0ĺr.t.
38
4
24
-
N-Me
0ĺr.t.
42
Table 3
P-OEt
Results of reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines with
furan and N-methylpyrrol via Scheme 12
a
yield are given after purification by column chromatography
Next, we considered the reaction of imines (7) and (8) with 1-phenyl-3methylpyrazol-5-on [Scheme 13, Table 4]. The reaction needed moderate heating to
60oC. The desired products (25 and 26) Į-aryl (hetaryl)-ȕ,ȕ,ȕ-trifluoroalanine were
obtained in good yield. Similar to above mentioned examples, aminoalkylation is
affected at the point of maximum ʌ-electron density in the system. The
19
F NMR and
P NMR signals were observed in a range typical for such compounds [§ δF: -76.3
31
(s, CF3)] and [§ δP: -0.7 (m, P(O)(OEt)2)]. The products were purified on silica gel
by flash column chromatography.
CF3
O
OEt
MeO2C
N
F3C
H3C
CO2Me
H3C
O
7
H
N
N
O
HN
O
X
Ph
N
CF3
Et2O, 60oC
O
N
O
N
O
O
Ph
OEt
MeO2C
H
25, 26
P
OEt
O
X= C or P-OEt
8
Scheme 13 Reactions of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines with
1-phenyl-3-methylpyrazol-5-on
- 40 -
Results and discussion
Besides we examined the reaction between imines and N,N-dimethylaniline
[Scheme 14, Table 4]. A nitrogen lone pair presence in the DMA molecule activates
very strongly leading to high reactivity towards different electrophiles such as imine
(7) and (8). The reactions were carried out at -40oC in anhydrous diethylether.
CF3
O
OEt
MeO2C
N
NMe2
O
O
CF3
7
O
Me2N
CF3
Et2O, -40oC
O
N
H
RT
CO2Me
X
O
OEt
MeO2C
27, 28
P
N
OEt
O
X= C or P-OEt
8
Scheme 14
Reactions of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines with
N,N-dimethylaniline
The corresponding products methyl 2-[4-(dimethylamino) phenyl]-N-(ethoxyoxalyl]
3,3,3-trifluoroalaninate
(27)
and
methyl
N-(diethylphosphonoformyl)-2-[4-
(dimethylamino)phenyl]-3,3,3-trifluoroalaninate (28) were obtained in good yield.
Reaction
Temperature,
o
C
Yield,
%
Entry
Product
R
X
Y
1
25
-
C
-
60
76
2
26
-
P-OEt
-
60
55
3
27
-
C
-
-40ĺr.t.
46
4
28
-
P-OEt
-
-40ĺr.t.
43
Table 4
Results of reaction between N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines
and 1-phenyl-3-methylpyrazol-5-on and N,N-dimethylaniline via Scheme 13 and 14
a
yield are given after purification by column chromatography
- 41 -
Results and discussion
Obviously, the typical electrophilic substitution on benzene rings bearing –NH2
or N(CH3)2 groups furnishes exclusively ortho- and para- substituents as a result of
the lone pair on the nitrogen which may participate in the resonance. The signals of
the
19
F NMR and
31
P NMR were observed in the range typical for such compounds
[§ δF: -71.3 (s, CF3)] and [§ δP: 3.8 (m, P(O)(OEt)2)].
However, in the case of the larger substituents like N(CH3)2, steric hindrance
is playing significant role and the electrophilic substituition in para position is the most
preferable position. In our experiment, we observed similar results; we got more para
product (C4-amidoalkylation). In the spectrum of the reaction mixture we were able to
identify signals belonging to the product in ortho position. We used flash column
chromatography on silica gel in order to purife it.
Our study of the reactions of methyltrifluoropyruvates imines with different
aromatic and heteroaromatic compounds led to the design of a general method for
trifluoroalkyl-Į-amino (amido) alkylation of aromatic system of the donor type. Above
described experiments indicate that in the aromatic rings the C-amidoalkylation had
been directed consistently and regioselectively to the sites of maximal density.
- 42 -
Results and discussion
3.1.4.2.1 X-ray crystallographic analysis of methyl N-[ethoxyoxalyl]-3,3,3trifluoro-2-(1H-indol-3-yl)alaninate
The structure of the compound (17) methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2(1H-indol-3-yl)alaninate was confirmed by X-ray data [Figure 13]. This compound
forms a triclinic crystal system with a space geometry P-1.
Figure 13 X-ray structure of the compound 17 methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(1H-indol-3yl)alaninate showing the atom-numbering scheme
It was found, that the planar conformation of (17) is stabilized by an intramolecular
hydrogen interactions between hydrogen from an amide N-H bond and oxygen O2
(2.117Å) [Figure 14].
- 43 -
Results and discussion
Figure 14 Another view on the compound 17
We found out that atom F2 from Tfm group is laying almost at the same plane with
carbon and nitrogen atoms from the indol ring [Figure 13].
o
Bond lenghts [pm]
Angles [ ]
O(4)- C(9)
121.30(6)
O(4)- C(9)- N(1)
127.80(5)
O(5)- C(10)
118.80(6)
O(4)- C(9)- C(10)
122.20(5)
C(7)- O(2)
120.10(7)
N(1)- C(9)- C(10)
110.00(4)
C(6)- F(2)
135.00(6)
O(5)- C(10)- O(6)
127.50(5)
O(5)- C(10)- C(9)
122.00(5)
O(6)- C(10)- C(9)
110.50(4)
Table 7 Selected geometric parameters for the compound 17
- 44 -
Results and discussion
3.1.4.2.2 X-ray crystallographic analysis of methyl N-[ethoxyoxalyl]-3,3,3trifluoro-2-(2-furyl)alaninate
The structure of the compound (21) methyl N-[ethoxyoxalyl]-3,3,3-trifluoro2-(2-furyl)alaninate was confirmed by X-ray crystallographic analysis. This compound
forms a triclinic crystal system with a space geometry P-1 [Figure 15].
Figure 15 X-ray structure of compound 21 methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(2-furyl)alaninate,
showing the atom-numbering scheme.
It was found, that the planar conformation of (21) is stabilized by a two-center
intramolecular hydrogen interactions between N-H amide bond and two oxygens:
O1 from furan and O5 from carbonyl group [Figure 16].
- 45 -
Results and discussion
C9
C5
N1
O1
H1
O1
C10
2.25 pm
O5
2.27 pm
Figure 16 The molecular structure presenting hydrogen bonds interactions
D-H…A
d(D-H)
d(H…A)
d(D…A)
<(DHA)
N(1)-H(1)…O(1)
82.9(18)
227.3(17)
267.31(19)
110.0(14)
N(1)-H(1)…O(5)
82.9(18)
225.1(17)
264.82(19)
109.7(14)
o
Table 6 Hydrogen bonds for 21 [pm and ]
o
Bond lenghts [pm]
Angles [ ]
O(4)- C(9)
120.20(17)
O(4)- C(9)- N(1)
126.31(12)
O(5)- C(10)
120.02(17)
O(4)- C(9)- C(10)
124.21(12)
N(1)- C(9)
135.20(17)
N(1)- C(9)- C(10)
109.48(11)
N(1)- H(1)
82.9018)
O(5)- C(10)- O(6)
126.78(12)
O(5)- C(10)- C(9)
121.80(12)
O(6)- C(10)- C(9)
111.41(11)
Table 7 Selected geometric parameters for the compounds 21
- 46 -
Results and discussion
3.1.4.3 Reduction
reaction
of
methyltrifluoropyruvates
(MTFP)
imines
containing N-oxalyl and N-phosphonoformyl groups with ʌ-donor
heteroaromatic compounds
Our further studies concerning chemistry of the new imines (7, 8) have shown
their very interesting nature. Taking into consideration the strong electrophilic nature
of the new imines bearing N-oxalyl and N-phosphonoformyl groups, we believed that
they can easily be reduced to the corresponding amines using commonly known
reducing agents. The reduction of the C=N double bond (C,N-Dihydro-addition) was
accomplished by the simple and convenient method using 1.5 equivalent of NaBH4
[Scheme 15] in anhydrous ether at room temperature. Under these conditions
N-oxalyl methyltrifluoropyruvate imine was reduced leading to the desired methyl
N-[ethoxyoxalyl]-3,3,3-trifluoroalaninate (29) in good 69% yield. Surprisingly, in the
case of the reduction reaction of N-phosphonoformyl methyltrifluoropyruvate imine
the unexpected azirine derivative was obtained in the preparative yield - methyl
3-(diethylphosphonyl)-2-(trifluoromethyl)-2H-azirine-2-carboxylate (30) [Scheme 15].
CF3
O
F3C
OEt
MeO2C
1.5 eq. NaBH4
Et2O, r.t.
N
O
H
OEt
MeO2C
N
H
O
O
7
CF3
29
O
OEt
MeO2C
N
O
F3C
P
OEt
P
1.5 eq. NaBH4
Et2O, r.t.
OEt
O
MeO2C
OEt
N
30
8
Scheme 15 Reduction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines with NaBH4
- 47 -
Results and discussion
The structure of azirine (30) was unambigously confirmed by NMR, IR and HRMS
data. We observed in
19
F NMR spectrum a single peak at -62.7 ppm belonging to
CF3 group. Furthermore, in
31
P NMR spectrum we observed a multiplet at -3.2 ppm
belonging to –P(O)(OEt)2 [Figure 17]. The IR spectra contained the streching
-62.711
vibration modes for P=O (1271 cm-1), C=O (1652 cm-1), C=N (1765 cm-1).
400
-3.461
-3.489
-3.384
-3.278
31P, CDCl3
-3.177
15000
-3.075
19F, CDCl3
300
10000
200
100
5000
0
0
-100
-50
-100
5.0
ppm (f1)
0.0
-5.0
-10.0
ppm (f1)
Figure 17
19
31
Spectra: F NMR (CDCl3) and P NMR (CDCl3) of the compound 30 methyl 3(diethylphosphonyl)-2-(trifluoromethyl)-2H-azirine-2-carboxylate
The positions of the carbon signals belonging to the azirine cycle also confirmed
the suggested structure: C-N 105.9 and 105.8 (both q, 2JCF = 40.9 Hz) and C=N
146.5 (d, 1JCP = 278.3 Hz).
3.1.4.4
Methyl
3-(diethylphosphonyl)-2-(trifluoro-methyl-2H-azirine-
2-carboxylate - formation and characteristic features
2H-Azirines are important class of compounds because of their versatile
chemical and biological behavior 94. An excellent example of this is fact, that they are
naturally occuring antibiotics
95,96
. In this context it should be also noted, that this
highly strained ring system is the smallest of the nitrogen-unsaturated heterocycles.
2H-Azirines possess high reactivity towards nucleophiles and electrophilic reagents,
- 48 -
Results and discussion
as well as in cycloadditions
97-99
. The utilization of the imines as substrates
in cycloaddition process is a direct way to different nitrogen-containing cyclic
molecules like for example azirines and aziridines
100
. Moreover, the ring opening
reactions both of azirine cycle and aziridine cycle represent a useful synthetic
pathway to a variety of interesting Į- and ȕ-amino acids 101.
Noteworthy, synthetic protocols of 2H-azirines, firstly reported by Neber et al
102,103
,
can be categorized into following classes 94:
ƒ
intramolecular reactions of N-functionalized imines, vinyl azides, isoxazoles,
and oxaazaphospholes [Figure 18, route a, b, c]
ƒ
intermolecular reactions between nitriles and carbenes and acetylenes
[Figure 18, route d and e]
N
LG
a
+
N
e
b
N3
N
d
c
+
N
P
Y
O
Y= C, P
Figure 18 Synthetic protocols for 2H-azirines
In our case, the mechanism of reaction of formation azirine (30) was not completely
clear. We assumed that the formation of azirine cycle was tentatively rationalized by
the tautomerization of the initially formed reduction product A to the enol B, followed
by dehydratation [Scheme 16].
- 49 -
Results and discussion
CF3
O
C
MeO2C
N
O
F3C
OEt
P
OEt
P
OEt
MeO2C
OEt
N
O
- H2O
NaBH4
CF3
O
H
F3C
C
OEt
N
H
MeO2C
P
C
C
MeO2C
OEt
OH
N
O
OEt
O
A
Scheme 16
OEt
P
B
Proposed mechanism of the formation of azirine cycle for the compound 30 methyl 3(diethylphosphonyl)-2-(trifluoromethyl)-2H-azirine-2-carboxylate
Interestingly, we found also that heating of the imine 8 (methyl 2-[(Ndiethylphosphonoformyl)] imino]-3,3,3-trifluoropropanoate), in - for example - dry
toluene yields to the respective aziridine derivative [Scheme 17].
CF3
O
O
F3C
OEt
P
OEt
MeO2C
N
P
MeO2C
toleune
OEt
O
OEt
N
30
8
Scheme 17
Transformation of imine 8 into azirine
(trifluoromethyl)-2H-azirine-2-carboxylate
30
methyl
3-(diethylphosphonyl)-2-
Indeed, continuing our library design concerning the synthesis of new derivatives of
Tfm amino acids, we attempted to open azirine cyclic of compound (30). Firstly, we
tried unsuccessfully the reaction of azirine with different organometallic reagents
(MeMgCl, PhMgCl). Then, we investigated to open azirine cyclic with mild
nucleophiles like furan. Much of our effords connected with the ring-opening
- 50 -
Results and discussion
reactions of the azririne cycle failed. Perhaps, the screening effects and size of these
three subsituents groups: CF3, CO2Me and –P(O)(OEt)2 on azirine cycle precluded
the attack of nucleophiles.
Despite of this, we were attempting to reduce azirine derivative to aziridine
compound. The reduction of C=N double bond in azirine cycle was accomplished
using 2 equivalent of NaBH4 [Scheme 18] in anhydrous ether at room temperature.
O
F3C
OEt
P
MeO2C
2eq. NaBH4
O
F3 C
OEt
P
OEt
Et2O, r.t.
MeO2C
N
N
H
30
OEt
83%
31
Scheme 18 Reduction of the azirine cycle into aziridine
Interestingly, according to recent data104, derivatives of 3-trifluoromethyl-aziridine
have become inhibitors of matrix metalloproteinases [Figure 19].
F3C
CONHON
activated 3-trifluoromethyl-aziridine-2-hydroxamates
N
MMP-3
MMP-9
OMe
Figure 19 Activated 3-trifluoromethyl-aziridine-2-hydroxamates- inhibitor of MMPs
The ring opening reactions of methyl 3-(diethylphosphonyl)-2-(trifluoromethyl)-2Hazirine-2-carboxylate
and
methyl
3-(diethylphosphonyl)-2-(trifluoromethyl)-2H-
aziridine-2-carboxylate are still under investigation in our laboratories.
- 51 -
Results and discussion
Hydrolysis of the ester group in Į-Tfm-Į-amino acids bearing
3.1.4.5
an N-oxalyl group
In order to receive free oxamic acids on the nitrogen atoms we followed a
convenient reaction with potasium hydrocarbonate
190
[Scheme 18, 19]. The
reactions were performed in the methanol/water mixture. The products (32) methyl Noxalyl-3,3,3-trifluoro-2-(5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylalaninate
acid and (33) methyl N-oxalyl-3,3,3-trifluoro-2-(5-methyl-3-oxo-2-phenyl-2,3-dihydro1H-pyrazol-4-yl)-alaninate acid were received in almost quantative yield.
F3C
1. KHCO3
MeOH/H2O (4:1)
r.t
O
Bz
O
MeO2C
F3C
OH
2. H+, H2O
N
H
O
Bz
MeO2C
N
H
O
O
93%
32
13
Scheme 18 Hydrolysis of the oxamate moiety into oxamic acid 32
F3C
CO2Me
F3C
H3C
O
HN
N
H
O
O
N
O
1. KHCO3
MeOH/H2O (4:1)
r.t
2. H+, H2O
Ph
CO2Me
H3C
OH
HN
N
H
O
N
93%
Ph
33
25
Scheme 19 Hydrolysis of the oxamate into oxamic acid 33
- 52 -
O
O
Results and discussion
3.2
Nitrogen-containing bisphosphonates (N-BPs) - potential
inhibitors of MMPs
This subchapter concerns designing and synthesis of a new class of
N-bisphosphonates. To the best of our knowledge 1,3 - dipolar cycloadition catalyzed
by Cu(I) giving the possibility to a wide range of novel nitrogen-bisphosphonates as
potent drug candidates, has not been yet applied in bisphosphonates chemistry.
Moreover, click methodology is giving the possibility to attach additional moiety to the
bisphosphonate molecule, namely a nitrogen-containing heterocycle - triazol.
3.2.1 Bisphosphonates (BPs) - general remarks
Bisphosphonates are structural analogues of inorganic pyrophosphate in
which an oxygen atom has been replaced by a carbon atom [Figure 20] 105-110.
O
O
OH
P
O
OH
P
OH
OH
P
OH
OH
P
OH
O
OH
O
Bisphosphonate
Pyrophosphate
Figure 20 Bisphosphonates as structuraly related to inorganic pyrophosphate
- 53 -
Results and discussion
Due to its ability to chelate metals, bisphosphonates were initially used either
as corrosion inhibitors or as complexing agents in textile, fertilizer or oil industries
In 1960s it was shown by Fleisch and co-workers
112
111
.
, that diphosphate can prevent
calcification by binding to newly forming crystals of hydroxyapatite (HA). Then, it was
found that bisphosphonates, similarly to PPi can prevent calcification both in vivo and
in vitro due to their high affinity towards hydroxyapatite. Most importantly,
bisphosphonates were shown to prevent pathological calcification in contrast to
pyrophosphonates 113.
Nowadays, bisphosphonates are well established as successful agents for the
prevention and treatment of diseases connected with calcium disorders like
postmenopausal osteoporosis
Paget’s disease
118,119
114-116
, corticosteroid-induced bone loss
117
and
. Interestingly, it was discovered that various matrix
metalloproteinases (MMPs) are inhibited in vitro by several bisphosphonates.
This novel finding may particularly explain the efficacy of bisphosphonates in their
current indications in human-beings
120,121
.
Furthermore, recently they have become
important class of compounds used in the management of cancer-induced bone
disease
122-124
. Finally, bisphosphonates are also of interest in the context of
immunotherapy as well as they possess potent effects against parasites responsible
for sleeping sickness, Chagas’ disease, malaria and leismaniases 125-127.
3.2.1.1 Chemical
structure
of
bisphosphonates.
Structure-Activity
relationships (SARS)
All bisphosphonates have the same generic structure. Even if their chemical
structure closely resembles the chemical structure of pyrophosphate (PPi), there are
many features, which differs them from each other. First of all, in PPi two phosphate
groups are linked by oxygen being hydrolitically unstable, whereas in BPs they are
linked to carbon, which render them hydrolitically stable and also to withstand
incubation in acids or with hydrolytic enzymes 128.
- 54 -
Results and discussion
The P-C-P moiety has been called the “bone hook”, namely it is the primary
structural feature, that endows the molecule its affinity and targets it to the bone
107
.
The “bone hook” is responsible for giving bisphosphonates high affinity to the bone,
which can further be enhanced by the substitution of a hydroxyl group in the side
chain R1. Interestingly, the side chain R2 plays an important role in determing the
potency of bisphosphonates [Figure 21] 107, 108.
When R1 is an OH, binding to
hydroxyapatie is enhanced
O
OH
R1
OH
P
C
P
OH
R2
OH
O
P-C-P "bone hook" is essential for
binding to hydroxyapatite
R2 side chain determines
potency
R2 = -CH3 (Editronate)
-CH2CH2NH2 (Pamidronate)
-CH2CH2CH2NH2 (Alendronate)
Figure 21 Structure-activity relationships in geminal bisphosphonates
It is noteworthy to add, that the presence of a hydroxyl group in the side chain R1 can
increase the ability of BPs to bind to bone mineral by preventing both crystal growth
and dissolution
129
. Moreover, its presence enhances the affinity for calcium and thus
bone mineral even further, owing to the ability of bisphosphonates to chelate calcium
ions.
Like PPi, bisphosphonates form a three dimensional structure capable of binding
divalent metal ions such as Ca2+, Mg2+, Fe2+ in the bidentate manner
appears figure [Figure 22] presenting this mechanism.
- 55 -
130,131
. Below
Results and discussion
2+
Figure 22 Mechanism of bisphosphonates to bind to divalent metal ions such as Ca
3.2.1.2 Nitrogen-containing bisphosphonates (N-BPs) - characteristic features
More recent studies have explored, that the structure present in R2 side chains
of bisphosphonates is the major determinant of antiresorptive potency. A turning
point concerning clinical investigation of bisphosphonates was found in BPs
containing an additional moiety – a nitrogen atom in the side chain R2 (N-BPs).
In particular, bisphosphonates such as Pamidronate or Alendronate with a basic
primary nitrogen atom were found to be up to 1000-fold more potent than nonaminobisphosphonates (Clodronate or Etidronate) [Figure 23]. Moreover, it was
determined that bisphosphonates containing a secondary amine group in the side
chain R2 are more potent up to 300-fold than those containing a primary amine
(Incadronate) [Figure 24]. Most importantly, the highest antiresorption potency has
been shown by bisphosphonates containing a tertiary nitrogen atom within ring
- 56 -
Results and discussion
structures
131
in the side chain R2. An example of this kind of N-BPs is Risendronate
and Zolendronate [Figure 23]. These cyclic nitrogen bisphosphonates has proved to
be up to 10.000-fold more active than for example Editronate (in experimental
systems used for human-beings)
107
. The difference in potencies might arise from the
different mechanisms of action exhibited by nitrogen containing bisphosphonates and
non-nitrogen containing bisphosphonates. Non-nitrogen containing bisphosphonates
are metabolically incorporated into adenosine triphosphate (ATP) producing
nonhydrolyzable ATP analogues. Nitrogen-containing bisphosphonates inhibit the
enzyme farnesyl diphosphate (FPP) synthase in the melavonate pathway 132.
The results obtained to date indicate that the key features required for high inhibitory
potency of N-BPs include:
ƒ
The presence of two geminal phosphonate groups responsible for interaction
with the molecular target
ƒ
The presence of a basic nitrogen in heterocyclic side chain affects potency
ƒ
The three-dimensional orientation of those basic nitrogen atom is critical for an
effective inhibition
ƒ
The geminal hydroxyl group does not influence the ability of the N-BPs to act
at the cellular level
ƒ
The introduction of lipophilic groups into N-BPs backbone can significantly
improve their pharmacokinetics increasing the availability for soft tissues.
Noteworthy, even if N-bisphosphonates differ from non-N-bisphosphonates with the
respect
to
antiresorptive
potency,
their
pharmacokinetics
are
characterized by highly selective localization and retention in bone 133.
- 57 -
similar
and
Results and discussion
O
O
O
OH
H
P
OH
HO
P
H
P
OH
P
Cl
P
Cl
P
OH
OH
OH
OH
H
OH
OH
OH
O
O
O
OH
OH
HO
P
H3C
P
OH
O
O
OH
P
HO
OH
OH
Cl
OH
OH
S
P
OH
OH
O
O
Figure 23 Non-nitrogen-BPs currently used in clinical setting
O
O
O
OH
HO
HO
P
P
H2N
O
O
HO
P
OH
OH
OH
N
P
OH
OH
N
P
OH
OH
N
HO
P
OH
OH
P
O
O
O
P
N
P
O
"
Figure 24 Nitrogen-BPs currently used in clinical setting
- 58 -
OH
OH
OH
O
!
OH
OH
O
O
HO
P
HO
OH
N
OH
P
O
OH
OH
P
OH
O
HO
P
O
O
N
N
OH
OH
OH
OH
OH
P
P
P
H
OH
OH
OH
H2N
OH
OH
OH
OH
Results and discussion
3.2.2 Synthesis of nitrogen-containing bisphosphonates (N-BPs) via click
methodology
3.2.2.1 Click chemistry - general remarks
The history of synthesis of natural products has been long and admirable.
However, drug discovery based on natural products is generally hampered by slow,
costly and complex syntheses
134
. The development of powerful, selective and
modular “blocks”, which work reliably in both small- and large-scale application has
always fascinated scientists
135
. A growing impact on this field has had bioconjugate
and combinatorial chemistry. Click chemistry, recently proposed by Sharpless et al.
135,136
is a modular approach that uses the most practical and reliable chemical
transformations:
“…the reaction must be modular, wide in scope, give very high yields, generate only
inoffensive byproducts that can be removed by no chromatographic methods and must be
stereospecific (but not necessarily enantioselective)…the requires process characteristics
include simple reaction conditions, readily available starting materials and reagents, the
use of no solvents or a solvents that is benign (such as water) or easily removed, and
simple product isolation…”.
Undoubtedly, click chemistry does not replace existing methods for drug
discovery, but rather, it complements and extends those
136
. Generally, click
chemistry bases on carbon-heteroatom bond-forming connection chemistry. Similarly
in connection to chemistry appearing in nature - all proteins arise from 20 building
blocks that are joined via reversible, heteroatom links-amides 136.
- 59 -
Results and discussion
Carbon-heteroatom bond forming reactions comprise the most common examples
including the following classes of chemical transformations 135:
ƒ
cycloadditions of unsaturated species, especially 1,3-dipolar cycloaddition
reactions, but also the Diels-Alder family of transformation
ƒ
nucleophilic substitution chemistry, particularly ring opening reactions of
strained heterocyclic electrophiles such as epoxides, aziridines, aziridinium
ions and episulfonium ions
ƒ
carbonyl chemistry of the “non-aldol” type, such as formation of the ureas,
thioureas, aromatic heterocycles, oxime ethers, hydrazones, and amides
ƒ
additions to carbon-carbon multiple bonds, especially oxidative cases such as
epoxidation, dihydroxylation, aziridination, but also Michael additions of Nu-H
To date, the most popular reaction, that has been adapted to fullfil these criteria is
the 1,3-dipolar cycloaddition
137
, also known as Huisgen cycloaddtion, between an
azide and a terminal alkyne affording the 1,2,3-triazole moiety
138
. Due to the fact
that azide and alkyne components can be incorporated into a wide range of
substituents, the potential of this reaction has become very high. Thus, the 1,3dipolar cycloaddition yielding triazoles is called “the cream of the crop” (it means the
best of a particular group) 135,139.
Most generally, Huisgen the 1,3-dipolar cycloaddition reaction introduced by Rolf
Huisgen
140
is an exergonic fusion process that unites two unsaturated reactants and
provides fast access to an enormous variety of five-membered heterocycles
141
.
A disadvantage of this method is fact that reactions usually afford mixture of the 1,4and 1,5-regioisomers [Scheme 20] 142,143:
- 60 -
Results and discussion
N
N
R1
N
N
R1
N
1
N
R1
N1
N
+
N
5
4
R2
R2
R2
ca. 1:1 mixture
Scheme 20 Products of thermal 1,3-cycloaddition
The lack of selectivity can be explained by the similarity in activation energies for
both processes
144
. Much of the effort for controlling this 1,4- versus 1,5-
regioselectivity failed. Furthermore, such intrinsic features made this reaction
unsuitable for being considered as “click reaction” 145.
The potential of this reaction has been recently enhanced by the discovery of
Sharpless et al. that copper(I) catalyzes [Scheme 21]
regioisomer of substituted 1,2,3- triazoles
147,148
146
the formation of a single
. Cu-catalyzed azide-alkyne 1, 3-
dipolar cycloaddition (CuAAC) has been established as one of the most reliable
means for the covalent assembly of complex molecules
149
. Instead of this, this
powerful bond forming process has proven to be extremely versatile, and has driven
the concept of “the click chemistry” from an ideal to a reality 150.
N
N
N
N
R1
Cu (I)
R1
N 1
N
4
R2
Scheme 21 Copper(I)-catalyzed synthesis of 1,4- disubstitued 1,2,3-triazoles
- 61 -
R2
Results and discussion
This process is strongly favoured in thermodynamic terms, yet both azides and
acetylenes are units, which are highly selective in their reactivity. Thanks to their
weak-base properties they are nearly inert toward biological molecules and toward
the reaction conditions found inside living cells151, 152. Additionally, they are showing
stability in range of different solvent, pH values and temperature.
3.2.2.2 Synthesis of mono and bispropargyl bisphosphonates
Our
first
efford
connected
with
designing
novel
nitrogen-containing
bisphosphonates (N-BPs) via click methodology was preparation of propargyl
substituted bisphosphonates, first unsaturated reactant. The synthesis of the
monopropropargyl bisphosphonates have been already reported by Yuan and Li
153
[Scheme 22, route A]. This procedure based on deprotonation of methylene
bisphosphonate (34) with NaH followed by alkylation with propargyl bromide. We
verified that this reaction is substantially accompanied by double alkylation (up to
35% of 38) leading to two products: tetraethyl but-3-yne-1,1-diyldiphosphonate (37)
and tetraethyl hepta-1,6-diyne-4,4-diyldiphosphonate (38) [Scheme 22, route A].
Furthermore, due to the similar Rf-values of these two products, their separation
using flash column chromatography was not enough effective in our experience
[Figure 25]. Unfortunately, much of our effort for achieving higher selectivity for
product (37) failed.
Consequently, we developed an alternative and easy method for the
preparation of (37) by selective addition of sodium acetylidene to our key
intermediate ethylidene bisphosphonate (36) [Scheme 22, route B] 154.
- 62 -
Results and discussion
O
O
1. NaH/ toleune
P
P
2.
P
OEt
Br
OEt
A
P
OEt
P
OEt
OEt
P
OEt
OEt
34
OEt
OEt
OEt
O
O
B
O
OEt
OEt
O
38
37
1. CH2O/ Et2NH, MeOH
2. cat-TsOH, tolene
O
O
OEt
OEt
P
OEt
OEt
P
P
Na
THF/ -20oC
P
OEt
OEt
OEt
OEt
O
O
37
36
Scheme 22 Synthesis of propargyl-substitued bisphosphonates
petroether: aceton
1:1
Rf = 0,40
Rf = 0,38
O
O
P OEt
OEt
P OEt
OEt
O
P OEt
OEt
P OEt
OEt
O
38
37
38 37
Figure 25 TLC presenting Rf values for compounds 37 and 38
Firstly, tetraethyl etylidenebisphosphonate (36) was prepared in multigram quantities
from tetraethyl methylene bisphosphonate (34) according to convenient and efficient
procedures given by Degenhardt et al
155
and Page et a
steps respectively from readily available starting materials.
- 63 -
156
[Scheme 23] via a two
Results and discussion
O
P
P
O
O
OEt
OEt
OEt
(CH2O)n , Et2N,
MeOH
P
MeO
H
OEt
O
P
A
OEt
OEt
OEt
P
P
OEt
O
34
cat. TsOH
toluene
B
OEt
OEt
OEt
OEt
O
36
35
Scheme 23 Synthesis of tetraethyl ethylidenebis (phosphonate) 36
Going
into
details,
treatment
of
methylene
bisphosphonate
(34)
with
paraformaldehyde and diethylamine in methanol is leading to ȕ-methoxyethylene-1,1bisphosphonate (35) [Scheme 26, route A], which can be very easily dehydrated by
an acid-catalyzed step elimination [Scheme 19, route B]. This reaction afforded the
desired compound (36) tetraethyl etylidenebisphosphonate in good overall yield
(89%). The product was used for the synthesis of mono-substituted bisphosphonate
[Scheme 25, route B].
Noteworthy, the reaction between tetraethyl etylidenebisphosphonate (36) and
sodium acetylidene gave excellent results even on a multi-gram scale (92%).
Moreover, the paramount advantage of this synthesis, in comparison to reaction with
NaH and propargyl bromide [Scheme 25, route B] is fact that it leads only to the
desired product (37). Besides, after ordinary work-up the product does not require
additional purification and can be directly used as a substract for click reaction.
Noteworthy, the synthesis of compound (38) - tetraethyl hepta-1,6-diyne-4,4diyldiphosphonate (the product of substantial double alkylation-Scheme 25) has been
reported already by Choi et al
157
. The alkylation of methylene bisphosphonate (34)
with two equivalents of propargyl bromide under deprotonation with two equivalents
of NaH in THF [Scheme 24] is a convenient and successful route leading to
dipropargyl derivative of bisphosphonates (38).
- 64 -
Results and discussion
2eq.
O
O
OEt
P
P
2eq. NaH
OEt
OEt
OEt
Br
P
THF/ r.t
P
OEt
OEt
OEt
OEt
O
O
38
34
Scheme 24 A convenient synthetic route to bispropargyl bisphosphonate 38
It found application in synthesis of ʌ-conjugated polymers due to expectation that
they can possess extremely large third-order optical susceptibility
158
. In particular,
poly (1,6-heptadiynes)s can be prepared by metathesis polymerization using
transition metal-catalysts [Scheme 25]. Obviously, the phosphoryl group (P=O)
provides a stronger coordinating site than the ether or carbonyl group and also
improves adhensive properties toward hard tissues 159,160.
O
O
EtO
EtO
P
P
O
O
P
P
EtO
OEt
EtO
Metathesis
OEt
OEt
OEt
Catalyst
*
*
n
Catalyst:
38
MoCl5, WCl6, PdCl2
Cocatalyst: (n-Bu)4Sn, EtAlCl2
Scheme 25 Cyclopolymerization of 38 by methathesis polymerization technique
- 65 -
Results and discussion
3.2.2.2.1
X-ray crystallographic
4,4-diyldiphosphonate
analysis
of
tetraethyl
hepta-1,6-diyne-
The structure of compound 38 was confirmed by X-ray analysis [Figure 26].
The central C4 atom is substituted with two propargyl groups and two phosphonate
groups. Molecules in the crystal has an approximately C2 symmetry. The single
crystal possesses a monoclinic system. The geometry around each P atom is that of
distorted tetrahedron, as can been seen from the ranges of bond angles around P1
and P2 of 100.66 (12) - 105.93 (13) and 100.89 (12) - 104.99 (13)o, respectively.
In each P(O)(OEt)2 group, the largest bond angle is that between the pair of non
ester O atoms.
Figure 26 X-ray structure of the compound 38, showing the atom-numbering scheme
- 66 -
Results and discussion
o
Bond lenghts [pm]
Angles [ ]
P(1)-C(4)
184.2(3)
O(1)-P(1)-O(3)
116.97(14)
P(2)-C(4)
183.7(3)
O(4)-P(2)-O(5)
117.52(16)
P(1)-O(1)
146.7(2)
O(1)-P(1)-O(2)
113.38(14)
P(2)-O(4)
146.6(2)
O(4)-P(2)-O(6)
113.16(16)
P(1)-O(3)
156.9(2)
O(3)-P(1)-O(2)
113.38(14)
P(2)-O(5)
157.2(3)
O(5)-P(2)-O(6)
113.16(16)
P(1)-O(2)
157.6(2)
O(1)-P(1)-C(4)
115.33(14)
P(2)- O(6)
158.2(2)
O(4)-P(2)-C(4)
115.39(16)
Table 8 Selected geometric parameters for the compound 38
3.2.2.3 Synthesis of azides (R-N3)
Since the discovery of organic azides by Peter Grieß more than 140 years
ago, numerous syntheses of these energy-rich molecules have been developed.
In more recent times in particular, completely new perspectives have been developed
for their use in peptide chemistry, combinatorial chemistry, and heterocyclic
synthesis. Organic azides have assumed an important position at the interface
between chemistry, biology and medicine 161.
A breakdown in triazole chemistry occurred with the observation by Sharpless’ group
concerning reaction between azides and terminal alkynes leading to triazoles 135.
- 67 -
Results and discussion
R
N
N
N
R
N
N
N
R
N
N
N
R
N
N
NH2
Figure 27 Resonance structures of azides R-N3
In principle, organic azides may be prepared through five different methods: insertion
of the N3 group by substitution of addition, insertion of an N2 group, insertion of
a nitrogen group, cleavage of the triazines and analogues compounds and finally by
rearrangement of azides 162.
In our experience, synthesis of aryl azides (39, 40) was prepared according to the
procedure proposed by Andersen et al. 163 [Scheme 26].
Br
N3
NaN3 (2 eq.), CuI (10 mol%)
ligand (30 mol%)
EtOH/ H2O (7:3)
95-100oC, 10h
39
Scheme 26 Synthesis of aryl azides
From the set of five ligands proposed by the author we have chosen ligand (N,Ndimethylethane-1,2-diamine), which efficiently accelerated the substitution of the
halide into azide moiety [Figure 28].
- 68 -
Results and discussion
OH
CO2H
N
H
N
H
N
H
MeHN
NHMe
MeHN
NHMe
Figure 28 The set of ligands used in the synthesis of aryl azides
Furthermore, we syntesized fluorinated azide, which could become very interesting
countepart of BPs in the 1,3-dipolar Cu(I) catalyzed cycloaddition by enriching the
acitivity of N-bisphosphonates. We prepared the new fluorinated azide 8-azido1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorooctane (41) from corrensponding 8-bromo1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorooctane according to the procedure proposed by
Wu et al. 164 [Scheme 27].
F
F
F
F
F
F
F
Br
F
F
F
F
F
F
NaN3, 18-C-6
F
F
F
F
F
N3
DMF, r.t.
F
F
F
F
F
F
F
F
41
Scheme 27 Synthesis of fluorinated azide
We found that synthesized by us azides are stable and can be purified by distillation.
- 69 -
Results and discussion
3.2.2.4 An active copper(I) catalyst
The conditions of click reactions since it has been discovered have varied
widely, especially due to the generation of the active species like for example Cu(I).
As mentioned before, Cu(I) catalysis dramatically improves regioselectivity to afford
the 1,4-disubstituted 1,2,3-triazoles. It is noteworthy to add, that in 2005, 1,3-dipolar
cycloaddition between alkynes and azides yielding only the 1,5-disubstituted triazoles
was reported 165,166. This click reaction was catalyzed by Cp*RuCl(PPh)3.
For the click reaction a various number of copper(I) sources can be utilized:
ƒ
Cu(I) salts, for instance CuI, CuBr 167
ƒ
in situ reduction of Cu(II) salts , for instance CuSO4
147,168
(reducing agents:
sodium ascorbate, hydroquinones and other quinones like Vitamin K
ƒ
comproportionation of Cu(0) and Cu(II) 146,169,170, e.g. excess of copper wire
ƒ
coordination complexes, like: [Cu(CH3CN)4]PF6
139
, (EtO)3P x CuI
171
,
[Cu(PPh3)3]Br 168,172
Interestingly, click reaction except of copper source does not require any special
chemical reagents. Generally, it proceeds to completion in 6 to 36 h at ambient
temperature in a variety of solvents, including aqueous tert-butanol or ethanol, and
very importantly water with no organic co-solvents
147
. Moreover, it tolerates a wide
range of pH values. Among several systems that have been developed for providing
click reactions, the most widely used are [Figure 28]:
- 70 -
Results and discussion
CuSO4 x 5H2O/ sodium ascorbate/ t-BuOH : H2O
CuSO4 x 5H2O/ sodium ascorbate/ CH2Cl2 : H2O
CuSO4 x 5H2O/ sodium ascorbate/ DMSO : H2O
CuI/ i-Pr2NEt/CH3CN, THF
Figure 29 Most common copper system used in click chemistry
For the synthesis of the novel nitrogen containing BPs we have applied two from the
above mentioned methods, namely:
ƒ
method A
CuI/i-Pr2NEt/CH3CN; in details copper(I) salt - CuI as a catalyst
in organic solvent (THF) and in the presence of as organic base (DIPEA)
ƒ
method B
CuSO4 x 5H2O/sodium ascorbate/t-BuOH : H2O; in details
generation of Cu(I) in situ from CuSO4 by presence of the reducing agent
(sodium ascorbate) in water-alcohol medium.
- 71 -
Results and discussion
3.2.2.5 1,3- Dipolar cycloaddition between monopropargyl bisphosphonate
and different azides
O
O
P
R
N3
P
OEt
OEt
OEt
OEt
P
Method A
P
N
Method B
R
N
N
OEt
OEt
OEt
OEt
O
O
Method A: CuI (10mol%), DIPEA (3eq), THF
Method B: CuSO4 (5mol%), Na-ascorbate (30mol%), t-BuOH/H2O (4:1)
Scheme 28 1,3-dipolar cycloaddition between monopropargyl bisphosphonates and different azides
We determined that Cu-catalyzed azide-alkyne 1,3-dipolar cycloaddition between
monopropargyl bisphosphonate and different azides [Scheme 28]: aryl azides [Table
9, entries 45 and 46], fluorinated azide [Table 4, entry 47], aliphatic azide [Table 4,
entry 48] proceeded smoothly at room temperature. Both above mentioned
methods: method A and method B are giving excellent results, however the method
B is most preferable due to a simpler isolation of the pure products, flash column
chromatography was not reguired.
Next, we have investigated the 1,3-dipolar cycloaddition between monopropargyl
bisphosphonate and highly functionalized azides [Table 9, entries 49 and 50]. We
discovered that 2,3,4,5-tetra-O-acetyl-ȕ-D-glucopyranosyl azide is an ideal precursor
for the synthesis of modified bisphosphonates. All the polysaccharides constitute
a class of natural compounds involved in a number of important biological functions.
Obviously, click reaction between them and monopropragyl bisphosphonates is
a simple and practical way to obtain bioactive compounds, potential candidates of
drugs 173,174. Moreover, we determined that 3’-azido-3’deoxythymidine (AZT) is a very
good partner for preparation of disubstituted 1,2,3 triazoles.
- 72 -
Results and discussion
ʋ
Product
R- N3
Method
Yield
A
79
B
87
A
73
B
80
A
81
B
85
B
72
B
68
O
OEt
P
OEt
N3
1
OEt
P
N
N
OEt
N
O
45
O
OEt
P
OEt
2
N3
OEt
P
N
N
OEt
N
O
46
O
OEt
P
OEt
OEt
P
3
N
N3
F13C6
N
C6F13
OEt
N
O
47
O
P
O
4
N3
P
N
O
OEt
OEt
O
O
N
N
OEt
OEt
O
48
O
P
OEt
OEt
AcO
AcO
5
AcO
AcO
O
O
N3
OAc
P
N
AcO
AcO
OAc
N
N
49
- 73 -
OEt
OEt
O
Results and discussion
O
O
N
6
NH
H
O
N
OH
O
N
O
OH
P
O
O
OEt
B
OEt
N3
P
N
N
N
92
OEt
OEt
O
50
Table 9 Reaction of monopropargyl bisphosphonate with different azides
In order to emphasize the importance of AZT, it is obligatory to mention that all of the
currently approved agents, which target HIV-rt (The Human Immunodeficiency Virus)
are dideoxynucleoside analogues
175-177
. Moreover, since the discovery of modified
acylnucleosides as antiviral agents, substantial efforts have been devoted to the
synthesis and biological evaluation of such compounds
178
. Indeed, click
methodology has opened up the possibility to design derivatives of AZT- potential
antiviral drug in the treatment of AIDS (Acquired Immune Deficiency Syndrome).
3.2.2.5.1 Catalytic cycle for the Cu(I) catalyzed 1,3-cycloaddition
Although the thermal dipolar cycloaddition of azides and alkynes occurs
through a concerted mechanism, DFT (the density functional theory) calculations on
monomeric copper acetylide complexes indicate, that the concerted mechanism is
strongly disfavoured relative to a stepwise mechanism 143.
In the system CuSO4 x 5H2O/sodium ascorbate, the active catalyst Cu(I) [Scheme 29,
moiety A] is generated in situ from CuSO4 via the reduction with sodium ascorbate
(reducing agents). Then the monopropargyl bisphosphonates [Scheme 32, molecule
1] coordinates with the Cu(I) by displacing one of the acetonitrile ligands [Scheme 29,
- 74 -
Results and discussion
moiety 1b, 2]. This kind of conversion of the alkyne to the acetylide is well involved in
many C-C bond forming reactions in which Cu acetylide species are bona fide
intermediates 169.
The formation of the copper acetylide was calculated to be exothermic
by 11.7 kcal/mol. This is consistent with well-known facility of this step, which
propably
occurs
through
the
intermediacy
of
ʌ-alkyne-copper
complex.
The ʌ coordination of an alkyne to copper is calculated to move the pKa of the
alkyne terminal proton down by ca. 10 units, bringing it into the proper range to be
proponated in an acqueous medium 149.
O
EtO
EtO P
EtO
P
EtO
O
LnCu
A
H
1
O
EtO
EtO P
EtO
P
EtO
O
CuLn-1
H
O
EtO
EtO P
EtO
P
EtO
O
1b
CuLn-1
2
Scheme 29 Generation of Cu(I) in situ from CuSO4x5H2O
Overall, few is known about the nature of the copper acetylide complexes active in
Cu(I)-catalyzed alkyne-azide coupling, even though evidence suggests, that these
species determine in large part the rate and success of catalysis.
Noteworthy, copper acetylide or cuprous acetylide is a typical multi-site bridging
ligand in carbonyl cluster derivatives
179
. Surprisingly, commercially available copper
acetylides, which are presumably already saturated with alkyne, show no catalytic
activity, emphasizing the importance of labile ligand dissociation to catalysis, and as
of yet, only one known copper species has been shown to catalyze triazole
formation 148,179.
- 75 -
Results and discussion
Following the formation of the active copper acetylide species [Scheme 30, molecule
2], azide replaces one of the ligands and binds to the copper atom via the nitrogen
proximal to carbon, forming intermediate 3 [Scheme 30]. The energy of chemical
individuum 3 approximates 0.0 kcal/ mol while the ligand exchange process 0.7 and
2.0 kcal/mol.
Next, the distal nitrogen of the azide attacks the C-2 carbon of the copper acetylide.
This leads to the formation of an unusual and intriguing six-membered copper
containg metallcycle 4. This step is endothermic by 12.6 kcal/mol with a calculated
barrier of 18.7 kcal/mol, which is considerably lower than the barrier for the
uncatalyzed reaction (approximately 26.0 kcal/mol). The energy barrier for the ring
contraction of 4, which leads to the triazolyl-copper derivative 5, is quite low
(3.2 kcal/mol). Proteolysis of 5 releases, the desired triazole product 6, thereby is
completing the catalytic cycle.
- 76 -
P
EtO
EtO
P
EtO
EtO
N
3
B-2
N
N
N
N
R2
R2
CuLn-2
4
N
CuLn-2
B-1
B-3
N
N
N
R2
P
EtO
EtO
O
P
O
N
EtO
EtO
O
P
P
5
N
N
2
R2
CuL n
CuLn-1
- 77 -
R2 = PhN 3, BzN 3, C2 H 4C 6F13, AZT, ... etc.
B - direct
EtO
EtO
Scheme 30 Proposed catalytic cycle for the Cu(I) catalyzed ligation
O
P
O
EtO
EtO
O
P
O
EtO
EtO
EtO
EtO
O
A
LnCu
C
O
P
P
O
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
O
P
P
O
N
6
1
N
N
H
R2
Results and discussion
Results and discussion
3.2.2.5.2 Triazoles - characteristic features
Triazoles represent a class of five membered heterocyclic compounds of two
carbon atoms and three nitrogen atoms. Recently, these kinds of heterocyclic
compounds have been shown to possess very interesting biological and medicinal
activity. Triazoles are especially relevant to drug discovery because of their
physicochemical properties discovery. They can mimic the atom placement and
electronic properties of peptide bond NH-CO [Figure 30]: 136,180,181
O
N
R2
R1
N
mimicked by
N
N
R2
R1
H
R1 to R2 distance
3.8 A
R1 to R2 distance
5.0 A
Figure 30 Topological and electronic similarities of amides and 1,2,3-triazoles
Most generally, triazoles serve as linking units that place the carbon atoms attached
to the 1,4 positions of the cyclic ring at a distance of a 5.0 Å (while C-Į distance in
amides: 3.8 Å). However, some structural differences between amides and triazoles
bond exist. In contrast to amides, triazoles cannot be cleaved hydrolytically or
otherwise, and unlike benzenoids and related aromatic heterocycles, they are almost
not capable to be reduced or oxidized. In spite of this, triazoles possess a much
stronger dipole moment than an amide bond, ~5 Debye (by ab initio calculation,
RHG/6-311G**; cf. N-methyl acetamide: 3.7-4.0 Debye. However, this chemical
features may here enhance peptide bond mimicry by increasing the hydrogen bond
donor and acceptor properties of the triazoles 143.
- 78 -
Results and discussion
In our experience 1,2,3- triazoles have been substituted with bisphosphonate group
at the position 4 and other moiety, like phenyl group at 1 position. Obviously, they
can constitute aza-analogue of Zolendronate, the most potent drug among
bisphosphonates to date. As already mentioned, bisphosphonates containing in R2
side chain a tertiary nitrogen atom within ring structures are characterized by the
highest antiresorption potency.
In order to obtain free N-BPs we have selected N-pivaloylmethyl derivative as the
most suitable precursor (sub-chapter 3.2.2.8).
3.2.2.6 Synthesis of bisphosphonates containing ditriazoles
Our further insight was gained by examination of the nature of dipropargyl
bisphosphonate
namely
tetraethyl
hepta-1,6-diyne-4,4-diyldiphosphonate
(38).
In particular, we were interested into synthesizing bistriazoles via “double-click
reaction”. In similar manner to copper catalyze 1,3-dipolar cycloaddition of monosubstituted propargyl bromide (subchapter 3.2.2.5), the reaction between dipropargyl
bisphosphonates was accomplished using method B [Scheme 31], which is more
preferable in terms of purification. Thus, we designed bisphosphonates bearing two
triazoles heterocycles to which different functionalities are attached, like benzyl,
fluorinated aliphatic chain or 2,3,4,5-tetra-O-acetyl-ȕ-D-glucopyranosyl moiety.
- 79 -
Results and discussion
EtO
EtO
N3
CuSO4 x 5H2O (20 mol%)
Na-ascorbate (10 mol%)
t-BuOH/H2O (4:1)
N
O
O
P
P
N
OEt
OEt
N
N
N
N
54
OAc
O
OEt
P
P
OEt
O
OAc
EtO
EtO
OAc
OEt
OEt
O
AcO
AcO
O
O
P
P
OEt
OEt
OAc
OAc
CuSO4 x 5H2O (20 mol%)
Na-ascorbate (10 mol%)
t-BuOH/H2O (4:1)
O
O
AcO
AcO
38
N
N
N
N
OAc
OAc
OAc
N
AcO
N
55
C6F13
EtO
EtO
N3
CuSO4 x 5H2O (20 mol%)
Na-ascorbate (10 mol%)
t-BuOH/H2O (4:1)
O
O
P
P
OEt
OEt
45
C6F13
N
N
N
N
N
C6F13
N
56
Scheme 31 1,3-Cycloaddition of dipropargyl bisphosphonate 38 with different azides yielding
bistriazoles
Copper catalyzed 1,3-dipolar cycloaddition between dialkynes and different azides, in
contrast to Cu (I) 1,3-dipolar cycloaddition between alkynes and different azides is
not common. However, it has found application for instance in bioconjugation field of
chemistry182 or in material science 183.
In our experience, reactions between one equivalent of tetraethyl hepta-1,6-diyne4,4-diyldiphosphonate and two equivalent of different azides were proceeded
smoothly at room temperature. Purification was greatly simplified due to the absence
of side products. The products (54, 55 and 56) were received in very good yields.
- 80 -
Results and discussion
3.2.2.6.1 X-ray crystallographic analysis of tetraethyl 2-(1-benzyl-1H-1,2,3triazol-4-yl)ethane-1,1-diyldiphoshonate
The structure of compound (54) was confirmed by X-ray data [Figure 31]. Molecules
in the crystal have an approximately C2 symmetry (a two-fold axis passess through
the C9 atom-atom CĮ). The single crystal possesses a triclinic system. In similarlity to
tetraethyl hepta-1,6-diyne-4,4-diyldiphosphonate (38), the geometry around each
P atom is that of distorted tetrahedron, as can be seen from the ranges of bond
angles around P1 and P2 of 102.54 (17) - 104.16 (16) and 102.50 (18) - 105.10 (2)o,
respectively. In each P(O)(OEt)2 group, the largest bond angle is that between the
pair of non ester O atoms.
Figure 31 X-ray structure of the compound 54 showing the atom-numbering scheme
- 81 -
Results and discussion
o
Bond lenghts [pm]
Angles [ ]
P(1)-C(9)
183.7(4)
O(1)- P(1)-O(3)
114.30(17)
P(2)- C(4)
183.7(4)
O(4)-P(2)-O(5)
115.00(18)
P(1)-O(1)
145.8(3)
O(1)-P(1)-O(2)
115.14(17)
P(2)-O(4)
145.6(3)
O(4)-P(2)-O(6)
113.50(3)
P(1)-O(3)
158.8(3)
O(3)-P(1)-O(2)
104.07(14)
P(2)-O(5)
156.4(3)
O(5)-P(2)-O(6)
104.60(3)
P(1)-O(2)
156.5(3)
O(1)-P(1)-C(9)
115.13(17)
P(2)-O(6)
152.4(4)
O(4)-P(2)-C(9)
114.86(16)
Table 10 Selected geometric parameters for the compound 54
3.2.2.7 Click chemistry in situ
Furthermore, we focused on the preparation of the N-bisphosphonates via
click chemistry in situ. Many 1,4-disubstituted 1,2,3-triazoles have been obtained
recently with excellent yields by a convenient one-pot procedure from a variety of
readily available aromatic and aliphatic halides without isolation of potentially
unstable organic azides intermediates 184. Although organic azides are generally safe
compounds, those of low molecular weight can be unstable and difficult to handle.
The paramount advantage of this method is that azide generated in situ does not
have to be isolated. The efficiency of in situ click chemistry has been already
demonstrated
by
the
discovery
of
novel,
acetylcholinesterase and carbonic anhydrase
- 82 -
highly
151,185-187
.
potent
inhibitors
of
Results and discussion
In our experiment, firstly we attempted to synthesize tetraethyl 2-(1-benzyl-1H1,2,3-triazol-4-yl)ethane-1,1-diyldiphosphonate (57), which was presented already in
section 3.2.2.5, via one pot reaction from corresponding benzyl azide. We found that
benzyl azide can be generated in situ from one equivalent of sodium azide and one
equivalent of benzyl bromide at room temperature in the solution of t-BuOH and
water (12h). This reaction was controlled by TLC. When the conversion of halide into
azide group was completed monopropargyl bisphosphonates was added. The
experiment was followed by addition of alkyne, source of Cu(I), namely method B
(CuSO4 x 5H2O) [Scheme 32].
O
O
P
P
O
Scheme 32
OEt
OEt
OEt
OEt
NaN3 ,
P
N3
P
CuSO4 x 5 H2O 20 mol%
Na ascorbate 10 mol %
t-BuOH/ H2O (4:1)
N
N
OEt
OEt
OEt
OEt
O
N
Synthesis of tetraethyl 2-(1-benzyl-1H-1,2,3-triazol-4-yl)ethane-1,1-diyldiphosphonate
57 via one pot reaction
Secondly, by using exactly the same approach we were successful to synthesize via
one pot reaction tetraphosphonates molecules [Scheme 33]. Going into details,
isomeric
bis(bromomethyl)benzenes
were
converted
in
situ
into
bis(azidomethyl)benzenes by the reaction with 2 eq. of sodium azide. Similarly to
above mentioned, after completed conversion of halides into azides, monopropargyl
azides, copper salt (II) and reducing agents were added (method B). Due to the
complexing ability of the methylenebisphosphonate moiety towards metals, under
these one-pot conditions tetraphosphonates (58, 59 and 60) - form strong chelates
with sodium bromide (the side product of the reaction). However, pure products can
be easily isolated by purification on column chromatography.
- 83 -
Results and discussion
NaN3 ,
N
Br
Br
N
N
N
N
CuSO4 x 5H2O (20 mol%)
Na-ascorbate (10 mol%)
t-BuOH/H2O (4:1)
(EtO)2P
(EtO)2P
P(OEt)2
O
x NaBr
N
P(OEt)2
O
O
O
58
NaN3 ,
O
P
OEt
OEt
P
OEt
OEt
O
37
Br
Br
N
N
N
N
N
CuSO4 x 5H2O (20 mol%)
Na-ascorbate (10 mol%)
t-BuOH/H2O (4:1)
(EtO)2P
(EtO)2P
P(OEt)2
O
F
x NaBr
N
O
O
59
P(OEt)2
O
F
F
F
F
F
NaN3 ,
Br
Br
F
N
F
N
N
CuSO4 x 5H2O (20 mol%)
Na-ascorbate (10 mol%)
t-BuOH/H2O (4:1)
N
N
(EtO)2P
O
(EtO)2P
P(OEt)2
O
x NaBr
N
60
O
P(OEt)2
O
Scheme 33 Synthesis of tetraphosphonates molecules via one pot reaction
3.2.2.8 Hydrolysis of the ester groups in nitrogen-bisphosphonates (N-BPs)
Hydrolysis of the ester groups at the phosphorus in bisphosphonates is
allowing receiving free bisphosphonic acids from which later pharmaceutically
acceptable salts can be easily prepared.
We followed standard procedures188,
189
. In details, by hydrolysis ester groups
of 1 eq. tetraethyl 2-[1-(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)-1H-1,2,3-triazol4-yl]ethane-1,1-diyldiphosphonate (47) with 7 eq. of trimethylsilyl bromide in
methanol, followed by treatment with acqueous solution of water we afforded 2-[1-
- 84 -
Results and discussion
(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)-1H-1,2,3-triazol-4-yl]ethane-1,1-diyl]bis
(phosphonic acid) (51) in a very good yield [Scheme 34].
O
O
P(OEt)2
P(OH)2
1. Me3SiBr/ CH3Cl
2. MeOH
r.t
N
C6F13
N
P(OEt)2
N
C6F13
N
N
P(OH)2
N
O
O
51
47
Scheme 34 Hydrolysis of the ester groups at the phosphorus atom
In order to obtain free N-Bisphosphonate, N-pivaloylmethyl derivative (48) was
selected as the most suitable precursor. We found that the pivaloylmethyl group
could be easily removed under basic conditions for a few minutes at room
temperature in methanol to afford compound (52) in a good yield [Scheme 38].
Next, hydrolysis of the ester groups at the phosphorus atom was quantatively
performed by treatment of (52) with trimethylsilyl bromide in chloroform followed by
treatment with aqueous methanol yielding (53) [Scheme 35].
- 85 -
Results and discussion
O
O
P(OEt)2
P(OEt)2
2% KOH/MeOH
O
N
O
N
20 min. 65%
P(OEt)2
N
H
N
P(OEt)2
N
N
O
O
52
48
1. Me3SiBr/ CH3Cl
2. MeOH
r.t
O
P(OH)2
N
H
N
P(OH)2
N
O
53
Scheme 35
Selective removing of pivaloylmethyl group from 52 followed by hydrolysis of the ester
groups at the phosphorus atom from 53
- 86 -
Results and discussion
3.3 Fluorinated bisphosphonates (FBPs) - potential inhibitors of
MMPs
The previous subsection was dedicated to the chemistry of new classes of Nbisphosphonates. This part will focus on the chemistry and investigation of a new
class of fluorinated bisphosphonates, and particularly on the facile synthetic route
leading to these derivatives. Both fluorine and phosphorus compounds play a vital
role in all living systems. We believed that designing compounds possessing these
two components can bring excellent results.
3.3.1 Fluorinated Bisphosphonates (FBPs) – general remarks
The introduction of fluorine or perfluoroalkyl groups into molecules
is a versatile tool for modifying their physicochemical properties and physiological
behavior by increasing lipophilicity. Moreover, the strong electron-withdrawing
inductive effect of fluorine can influence the reactivity of neighbouring groups, for
example, acidity 3,63,191.
In spite of this, unlike other substituents, fluorine does not introduce large
steric perturbations and imparts increased hydrolytic stability as well as oxygen
solubility
192
Most importantly, mono- and difluoromethylene bisphosphonates have
been shown to exhibit interesting biological properties. They are potential candidates
for phosphate analogues 193,194 and inhibit bone lysis 195,196.
Moreover, difluoromethylidene bisphosphonates (F2MBP) can increase
alkaline phosphatase activity and fatty acid oxidation in calvaria cells in culture
Most importantly, it was demonstrated by Pietrzyk et al.
198
197
.
that F2MBP inhibits bone
resorption both in vitro and in vivo. Above presented results emphasize diversity
of fluorinated bisphosphonates as potential inhibitors of MMPs.
- 87 -
Results and discussion
It is noteworthy to add, that the synthetic application of fluorine-containing
analogs suffers from the pauticity of simple and effective methods for prepartion of
such compounds. Recently, Röschenthaler et al.
convenient
pathway
to
F3-Etidronic
acid
191
, were capable to present a
(1-hydroxy-2,2,2-trifluoroethylidene-
bisphosphonate)- fluorinated analogue of the commonly known Etidtronate [Figure
32].
O
O
OH
HO
P
H3C
P
OH
OH
OH
HO
P
F3C
P
OH
OH
OH
OH
O
O
Editronic acid
F3-Editronic acid
Figure 32 F3-Etidronic acid - fluorinated analogue of Etidronate
In their case, the reaction of trifluoroacetyl chloride and tris(trimethylsilyl) phosphite 2
at -70oC furnished chlorotrimethylsilane and 1-trimethylsiloxy-2,2,2-trifluoroethanetetrakis(trimethylsilyl)bisphosphonates 3 (F3-pentakis(trimethylsilyl)etidronate). The
bisphosphonates was then easily converted into free acid using a methanol/water
mixture [Scheme 36].
- 88 -
Results and discussion
O
OSiMe3
O
F3 C
C
Cl
P(OSiMe3)3
2
O
O
P(OSiMe3)3
2
OSiMe3
F3C
C
P
1
P
F3C
OSiMe3
3
C
P
OSiMe3
OSiMe3
OSiMe3
OSiMe3
O
4
O
OH
P
HO
MeOH/H2O
C
F3C
P
OH
OH
OH
O
5
Scheme 36 Synthesis of 1-hydroxy-2,2,2-trifluoroethylidene-bisphosphonic acid
The reaction of phosphorus(III) nucleophiles (trialkyl phosphites) with Į-halocarbonyl
compounds is well recognized in organophosphorus chemistry. It can result in
oxophosphonates (the Arbuzov reaction) and/or in enol phosphates (the Perkow
reaction) 199,200 [Scheme 37, 38].
OR1
OR1
O
R2X
R1O
P
R1O
P
R2
heat
R1O
P
-R1X
OR1
OR1
Scheme 37 The Michealis-Arbuzov reaction
X
201, 202
- 89 -
OR1
R2
Results and discussion
O
OR1
R2
R1 O
C
P
R1O
OR1
R2
heat
R3
P
CH
R1 X
C
R1 O
X
Scheme 38 The Perkow reaction
O
CH
X = Cl, Br
R3
203
Both reactions are of a very big scope and they found application in many different
synthesis.
For instance, Ishihara et al.
204-206
in their continous studies concerning synthesis of
fluorinated vinylic compounds bearing hetero-functional group, developed efficient
method for the synthesis of 1H-F-1-alkene-1-phosphonates (3) from F-alkanoic acid
chlorides (2) and triethylphosphite (1) [Scheme 39].
O
O
P(OR)3
RfF2C
C
Cl
O
Rf
C
0oC- r.t.
P
OR
C
F
OR
P
OR
H
Rf
BuLi-CuLi
C
C
-78oC, 15 min
F
P
OR
1
Rf = CF3, C2F5, n-C6F13
2
O
OR
OR
3
O
R = Et
Scheme 39 Synthesis of F-1-alkene-1-phosphonate
3.3.2 A facile synthetic way to difluoro(phosphonooxy)vinylphosphonate
In relation to above presented chemistry, we investigated a synthetic
pathway to a fluorinated derivative of vinylidene bisphosphonates difluoro
(phosphonooxy)vinyliphosphonate [Scheme 40]. We found that 2 eq. of phosphite
- 90 -
Results and discussion
esters both: tris(trimethylsilyl) phosphite and triethylphosphite reacted readily with
1 eq. 2-chloro-2,2,-difluoroacetyl chloride in dry monoglyme (7a) or dry THF (7b)
at -20 oC to room temperature. The progress of the reaction was controlled by
NMR and
31
19
F
P NMR. When the conversion was completed, the solvent was
evaporated in vacuum and the residue was purified by distillation. The products were
received in a very good yield. Difluoro (phosphonooxy)vinyliphosphonates are stable
in a dry inert atmosphere.
O
O
O
monoglyme
XF2C
C
Cl
F 2C
P(OR)3
C
o
-20 C to RT, 4h
1eq
P
OR
OR
OR
P
OR
2eq
O
R= SiMe3, Et
X= Cl
61
62
(R= SiMe ) 85%
(R= Et)
74%
Scheme 40 Synthetic route to difluoro(phosphonooxy)vinylphosphonate 61 and 62
The structures of compound (61 and 62) were confirmed by NMR analysis.
Below appears a
19
F NMR and
31
P NMR schemes presenting signals belonging to
fluor and phosphor atoms for the compound (61).
-94.00
-94.10
-94.20
ppm (t1)
Figure 33
-94.30
-94.40
-94.60
-94.70
-79.50
ppm (t1)
19
NMR spectra of compound 61, signals due to two fluor nuclei
- 91 -
-80.00
-80.046
-80.012
-79.915
-79.880
-79.747
-94.50
-79.781
F (188.31 MHz, CDCl3)
-94.459
-94.413
-94.365
-94.320
-94.275
-94.227
-94.181
19
Results and discussion
Both fluor atoms F1 and F2 are represented by multiplets: doublet of doublet of
doublet (ddd). Interestingly, except of coupling constants between fluor atoms 2JFF
we were also able to calculate followed coupling constants: 3JPF in both positions cis
and trans and 4JPF in both positions cis and trans.
4
JPFcis
4
O
2
JFF
JPFtrans
O
OSiMe3
F1
F
O
P
P
OSiMe3
OSiMe3
F
OSiMe3
2
3
JPFtrans
P
P
OSiMe3
JFF
F2
OSiMe3
O
OSiMe3
3
JPFcis
O
δ: -94.15 (ddd, 1F)
OSiMe3
O
δ: -79.62 (ddd, 1F)
2
2
3
3
JFF
= 26.0 Hz
JPFtrans = 17.5 Hz
4
JPFcis = 8.9 Hz
JFF = 25.0 Hz
JPFcis = 6.5 Hz
4
JPFtrans= 6.5 Hz
In analogy to fluor atoms, two phosphonate groups are also represented by multiplets
ddd. Similar, we were able to calculate identical coupling constants 3JPF and 4JPF.
-12.00
-12.50
ppm (t1)
Figure 34
-13.00
-13.50
-20.00
-20.50
-20.802
-20.722
-20.616
-20.535
P (80.99 MHz, CDCl3)
-13.098
-13.018
-12.877
-12.799
-12.721
-12.580
-12.499
31
-21.00
ppm (t1)
31
NMR spectra of compound 61, signals due to two phosphorus nuclei
- 92 -
-21.50
Results and discussion
4
3
JPFcis
O
JPFtrans
O
OSiMe3
F
O
P
F
3
JPFcis
3
P
JPP
F
OSiMe3
OSiMe3
F
OSiMe3
4
O
δ: -12.71
JPFtrans
OSiMe3
O
P
P
OSiMe3
OSiMe3
OSiMe3
O
δ: -20.71
3
4
JPFcis = 6.5 Hz
3
JPFtrans= 17.7 Hz
4
We found that 3JPFcis < 3JPFtrans and
4
JPFcis = 8.7 Hz
JPFtrans = 6.5 Hz
JPFcis > 4JPFt
Obviously, in the analogues compound (62), constant coupling calculated for fluor
and phosphor atoms of tetraethyl 3,3-difluoroprop-2-en-1,2-diylbis(phosphonate) are
-92.00
ppm (f1)
Figure 35
-92.50
-80.20
-80.30
-80.40
-80.50
ppm (f1)
19
NMR spectra of compound 62, signals due to two fluor nuclei
- 93 -
-80.60
-80.709
-80.676
-80.594
-80.559
-80.478
-80.445
-92.234
-92.189
-92.140
-92.094
-92.042
-91.997
comparable to (61).
-80.70
-80.80
-80.90
-81.00
10.0
5.0
0.0
ppm (t1)
ppm (t1)
Figure 36
-1.0
-2.0
-3.207
-3.144
5.996
6.097
6.629
6.574
6.519
6.467
6.413
6.363
6.313
6.260
6.208
6.153
6.731
Results and discussion
-3.0
-4.0
-5.0
-6.0
-7.0
31
NMR spectra of compound 62, signals due to two phosphorus nuclei
4
JPFcis
4
O
JPFtrans
O
OEt
2
F
O
F
P
JFF
P
OEt
OEt
2
OEt
JPFtrans
O
F
P
JFF
OEt
3
F
JFF
3
JPFtrans
4
JPFcis
OEt
OEt
OEt
3
JPFcis
O
δ: -92.14
2
P
O
δ: -76.90
2
= 26.6 Hz
= 18.0 Hz
= 8.6 Hz
JFF
= 25.0 Hz
JPFcis = 15.0Hz
4
JPftrans = 4.5 Hz
3
3.3.3 Studies concerning the reaction mechanism
The precursors initially react in a Michaelis-Arbuzov manner to give
(chlorodifluoroacetyl)phosphonate, with a subsequent Perkow reaction. Our results
are of interest since reaction of trialkyl phosphite with ketones is known to give the
Arbuzov reaction product in preference to the Perkow reaction product while the
- 94 -
Results and discussion
reaction of Į-chloroketones usually gives the exclusively formation of the Perkow
reaction products.
Generally, in the reaction 1 eq. of Į-halo ketones with 1 eq. of trialkyl phosphite to
produce an alkyl phosphonate, the first step involves a nucleophilic attack of the
phosphorus (in our case tris(trimethylsilyl) phosphite) on the alkyl halide (in our case
2-chloro-2,2,-difluoroacetyl chloride) [Scheme 41]. Dealkylation of the halide ion
results in the formation of the trialkoxyphosphonium salt and subsequently
a phosphoryl bond. This part of the reaction mechanism is referred to the MichaelisArbuzov pathway. Furthermore, the carbonyl addition reaction of the second eq.
of tris(trimethylsilyl) phosphite affords the product of a Perkow reaction (64). Going
into details, the initially formed intermediate due to the rapid intramolecular
rearrangment of a trimethylsilyl group, is transfered into a three-membered transition
state. Consequently, the Perkow reaction product containing unsaturatated systems
appears as a result of the predominant cleavage of the P-C bond of the the
intermediate. Taking into consideration the fact that –P(O)(OSiMe3)2 is an electronwithdrawing group, the P-C bond of the transition state is easily cleaved because the
carboanion formed as a result of the cleavage can be stabilized through
delocalization of electrons.
- 95 -
Results and discussion
SiMe3
Cl
O
ClF2C
C
:P(OSiMe3)3
Cl
ClF2C
O
O
C
P
OSiMe3
OSiMe3
-Me3SiCl
ClF2C
Me3SiO
Me3SiO
O
OSiMe3
C
P
P
O
O
C
P
OSiMe3
OSiMe3
XF2C
:P(OSiMe3)3
OSiMe3
OSiMe3
O
O
Cl
F2 C
Me3SiO
Me3SiO
OSiMe3
O
C
P
O
P
O
OSiMe3
-Me3SiCl
OSiMe3
F2C
OSiMe3
C
P
SiMe3
P
OSiMe3
OSiMe3
O
O
61
Scheme 41 Reaction mechanism (Perkow/Arbuzov)
Our results described above suggest the interesting evidence for the initial attack of
the phosphite molecule on the carbonyl carbon in a Perkow reaction.
3.3.4 Hydrolysis of the ester groups on phosphorus atom
In order to receive free acid 3,3-difluoroprop-2-en-1,2-diyldiphosphonic acid
(63) we hydrolysed trismethyl silyl groups on phosphorus atoms using standard
procedure
195
. Going into details, difluoro (phosphonooxy)vinyliphosphonate was
easily converted into the free acid using a methanol/water mixture [Scheme 42].
- 96 -
Results and discussion
O
O
F 2C
C
P
P
O
OSiMe3
OSiMe3
O
MeOH/H2O
F2C
OSiMe3
C
P
OSiMe3
O
P
OH
OH
OH
OH
O
63
61
Scheme 42 Conversion of the ester into free acid 63
Interestingly, we verified that this method of hydrolysis is not accompanied by the
cleavage of the C-O-P(O)(OEt)2 bond, as we could expect. The product was received
in a good yield.
- 97 -
Experimental section
Chapter 4
Experimental section
4.1 General methods
All the reactions were performed at glass equipment Duran 50.
All solvents used in reactions were freshly distilled from approximate drying agents
before use:
ƒ
diethyl ether and THF were dried over metallic sodium
ƒ
toluene was dried over CaH or metallic sodium
ƒ
petroleum ether was dried over Ca(OH)2
ƒ
methanol was dried over Ca(OH)2
ƒ
chloroform was dried over CaH2
ƒ
monoglyme was dried over metallic sodium
All other agents were recrystallized or distilled when necessary.
Syntheses of derivatives of Į-CF3-Į-amino acids possessing N-oxalyl and Nphosphonoformyl groups were performed under an atmosphere of dry nitrogen.
Syntheses
of
mono-
and
bispropargyl
bisphosphonates
and
ethylidenebisphosphonate were performed under an atmosphere of dry nitrogen.
Products were purified using flash column chromatography. Column chromatography
was performed using two types of silica gel:
- 98 -
Experimental section
ƒ
Silica gel 60 (0.2-0.5 mm) - Merck
ƒ
MP Silica 32-63, 60Å - MP Biomedicals
As an eluent were used followed mixtures of solvents:
ƒ
Ethyl acetate/light petroleum ether
ƒ
Ethyl acetate/chloroform
ƒ
Ethyl acetate/hexane
ƒ
Ethyl acetate/ethanol
ƒ
Acetone/ cyclohexane
The Rf values were determined on analytical TLCs plates – Silica gel cards, 60 F254,
0.20 mm-Merck. Visualization was accomplished by UV light or spraying by mixture
of Ce(SO4)2 x 4H2O and (NH4)6Mo7O24 x 4H2O in 5% of H2SO4.
4.2 Physical methods
Melting points and pressures are uncorrected. Melting points were determined with
an Electrothermal IA9100 digital melting point apparatus.
NMR spectra were recorded on Bruker DPX- 200 (1H NMR, 200.13 MHz,
50.32 MHz,
1
19
F NMR, 80.99 MHz,
( H NMR, 300.13 MHz,
13
31
13
C NMR,
P NMR, 188.31 MHz) and Bruker Avance- 300
C NMR, 75.47 MHz,
31
P NMR, 121.49 MHz) using the
residual proton signals of the deuterated solvent as an internal standard (1H and
13
C)
relative to TMS, H3PO4 (31P), CFCl3 (19F) as external standards.
Signals are described as follows: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet).
- 99 -
Experimental section
Mass spectra and high-resolution mass spectra (HRMS) were recorded on a Varian
MAT CH 7A at 70 eV.
Infrared spectra (IR) were recorded using a thin layer of the sample on a Fouriertransform “Magna-IR” (Nicolet) spectrometer (resolution 2cm -1, 128 scans).
X-ray structural study was carried out on a Siemens P4- four-circle or a Stoe IPDS
diffractrometer using graphite- monochromated Mo KĮ (Ȝ = 71.073 pm). All nonhydrogen atoms were refined with anisotropic thermal parameters. The hydrogen
atoms were refined with a riding model and mutual isotropic thermal parameters.
4.3 Synthesis and analytical data
Methyl 3,3,3-trifluoropyruvate (MTFP) (2)
was obtained via the references (85-87)
ƒ
Methyl 2,3,3,3-tetrafluoro-2-methoxypropanoate 1a
The reaction flask was charged with methanol (1.75 mL; 1383.7 g; 43.1 mol) and
heated. Then hexafluoropropene oxide HFPO (1) was added into refluxing methanol
at such a rate that almost all gase was consumed. During the period of 10 h, 325 g (2
mol) of HFPO was condensed. Then, flask with reaction mixture was cooled down till
50oC and washed with water §5 L. The organic layer was separated from water layer
and dried over CaCl2. Methoxypropanoate (485 g) received by methanolysis of HFPO
was used for further transformations.
- 100 -
Experimental section
ƒ
Methyl 3,3,3-trifluoropyruvate (MTFP)
A round-bottomed flask equipped with condenser was charged with methyl 2,3,3,3tetrafluoro-2-methoxypropanoate 48.5 g (255.4 mmol), SiO2 (3.5g; 58.3 mmol) and
concentrated sulfuric 95% acid (50 mL). The reaction mixture was stirred vigorously
and heated over a period of 2h. The product was purified by distillation
Yield 86% light yellow liquid, B.p. 85-92 oC/0.5 Torr
1
H NMR (200.13 MHz, CDCl3) δ: 3.90 (s, OCH3, 3H)
19
F NMR (188.31 MHz, CDCl3) δ: -71.7 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 55.2 (s, OCH3), 121.7 (q, CF3, 1JCF= 288.2 Hz),
156.3 (m, COOCH3), 162.1 (s, CO-CO)
Diethylcarbamoylphosphonate (4)
was obtained via references (39, 40)
Ethyl chloroformate (5.5 g; 50 mmol) was dissolved in dry THF. The reaction mixture
was heated up to 70oC. Then, triethylphosphite (8.4 g; 50 mmol) was added
dropwise. After a period of 1h, the reaction mixture was cooled down up to -30oC and
ammoniac (150 mmol) was condensed. Next, the temperature of the reaction mixture
was allowed to rise slowly up to room temperature. The product was filtered off from
the residue. Carbamoylphosphonate was recrystallized from THF.
Yield 86%, white solid, mp 126-128oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.31 (t, CH3, 6H, 3JHH= 7.2 Hz), 4.20 (m, OCH2, 4H),
7.32 (s, NH, 1H)
- 101 -
Experimental section
31
P NMR (80.99 MHz, CDCl3) δ: -0.5 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.5 and 16.6 (d, CH3, 3JCP= 6.1Hz), 65.6 and 65.7
(d, OCH2), 170.2 (m, CO)
General procedure for preparation of hemiaamidals (5 and 6)
Methyltrifluoropyruvate (2) (MTFP; 10 mmol) was added to ethyloxamate (3) or
diethyl carbamoylphosphonate (4); 10mmol. The reaction mixture was kept at room
temperature for 16h. Then, the crude solid was washed with petroleum ether to give
analytically pure hemiamidals.
2-(Ethoxyoxalyl-amino)-3,3,3- trifluoro-2-hydroxy-propionic acid methyl ester
(5)
Yield 98%, white solid, mp 58-61oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.42 (t, CH3, 3H, 3JHH= 7.1 Hz), 3.90 (s, OCH3, 3H),
4.41 (q, OCH2, 2H, 3JHH= 7.1 Hz), 5.42 (s, OH, 1H), 8.11 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -81.7 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 16.6 (s, CH3), 55.2 (s, OCH3), 65.5 (s, OCH2), 80.5
(q, Cα, 2JCF= 29.5 Hz), 121.6 (q, CF3, 1JCF= 285.O Hz), 156.1 (d, NH-CO), 158.3 (s,
CO-CO), 159.1 (m, COOCH3)
HRMS calculated for C8H10F3NO6 (M+) 273.0460, found 273.0462.
- 102 -
Experimental section
2-(Diethylphosphonoformamido)-3,3,3-trifluoro-2-propionic acid methyl ester
(6)
Yield 97%, white solid, mp 69-72oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.4 (t, CH3, 6H, 3JHH= 7.0 Hz), 3.93 (s, OCH3, 3H),
4.20 (m, OCH2, 4H), 5.32 (s, OH, 1H), 8.51 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -75.5 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -1.9 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.6 and 16.7 (d, CH3, 3JCP= 5.9Hz), 55.3 (s, OCH3),
65.6 and 65.7 (d, OCH2), 80.5 and 80.6 (both q, Cα) 2JCF= 29.0 Hz), 122.5 and 122.6
(both q, CF3, 1JCF= 286.O Hz), 165.5 (d, 1JCP= 123.0), 170.2 (m, CO)
HRMS calculated for C9H15F3NO7P (M+) 337.0538, found 337.0539
General procedure for preparation of imines (7 and 8)
A hemiamidal (29 mmol) was dissolved in dry ether (100 mL) and stirred vigorously.
To this mixture trifluoroacetic acid anhydride (4.5 mL, 31.9 mmol) was added
dropwise over a period of 0.5 h. Then, pyridine (5.2 mL, 64.0 mmol), was added
slowly. The reaction mixture was cooled down to -20oC and the precipitated
pyridinium trifluoroacetate was filtered off under an inert gas atmosphere. The filtrate
was concentrated in vacuum and triturated with petroleum ether (3 x 100mL) to
dissolve the imine and separate it from residual pyridinium trifluoroacetate.
The combined petroleum ether solutions were evaporated. Imines were additionally
purified by distillation. Analytical sample of imine (8) was not obtained. Imine (8)
proved to be unstable under distillation conditions; therefore it was used further
as a crude product (purity ca. 90% according to the NMR data).
- 103 -
Experimental section
Methyl 2[N-(2-ethoxyoxalyl)imino]-3,3,3-trifluoropropanoate (7)
Yield 82% colorless liquid, B.p. 95-97 oC/0.5 Torr.
1
H NMR (200.13 MHz, CDCl3) δ: 1.42 (t, CH3, 3H, 3JHH = 6.9 Hz), 4.08 (s, OCH3, 3H),
4.41 (q, OCH2, 2H, 3JHH = 6.9 Hz)
19
F NMR (188.31 MHz, CDCl3) δ: -71.3 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 14.2 (s, CH3), 55.6 (s, OCH3), 64.4 (s, OCH2), 122.6
(q, CF3, 1JCF = 279.0 Hz), 154.7 (m, CO), 156.3 (q, Cα, 2JCF = 35.1 Hz), 160.4 (m,
COOCH3), 167.3 (m, N-CO)
IR (thin layer) ν/cm-1: 1034 (C-O-C), 1638 (C=N), 1756, 1760 and 1765 (C=O)
HRMS calculated for C8H8F3NO5 (M+) 255.0457, found 255.0461
Methyl 2-[(N-diethylphosphonoformyl)] imino]-3,3,3-trifluoropropanoate (8)
Yield 75 %, pale yellow oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.69 (t, CH3, 6H, 3JHH = 6.8 Hz), 4.07 (s, OCH3, 3H),
4.33 (m, OCH2, 4H)
19
F NMR (188.31 MHz, CDCl3) δ: -71.3 (s, CF3
31
P NMR (80.99 MHz, CDCl3) δ: -2.2 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.0 and 16.3 (d, CH3, 3JCP=5.9Hz), 55.3 (s, OCH3),
63.2 and 63.5 (d, OCH2), 122.9 and 123.2 (both q, CF3, 1JCF = 282.0 Hz), 151.2 (m,
COOCH3), 155.1 and 155.4 (both q, Cα, 2JCF = 33.0 Hz), 164.8 (d, 1JCP = 120.0 Hz),
168.2 (dq, CO)
IR (thin layer) ν/cm-1: 1022, 1046 (P-O-C, C-O-C), 1270 (P=O), 1651 (C=N), 1759
and 1769 (C=O)
- 104 -
Experimental section
General procedure for the preparation of compounds 9 - 16
A Grignard reagent (solution in THF, 10.0 mmol) was added dropwise to a stirred
solution of an imine (10.0 mmol) in dry THF (25 mL) at -78 oC. After 1 h at -78 oC the
reaction mixture was allowed to warm up to room temperature within 2 h. The
reaction mixture was quenched with 1N HCl and extracted with ether (2 x 25 mL).
The combined organic layers were washed with brine (25 mL), dried over MgSO4 and
filtered. The solvent was removed under reduced pressure and the crude product
was purified by flash chromatography on silica gel (eluent: ethyl acetate/hexanes).
Methyl 2-{N-(2-ethoxyoxalyl)amino}-3,3,3-trifluoro-2-methylpropanoate (9)
Yield 65%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.43 (t, CH3, 3H, 3JHH = 7.2 Hz), 1.82 (s, CH3, 3H),
3.81 (s, OCH3, 3H), 4.44 (m, OCH2, 2H), 7.70 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -77.3 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 14.2 (s, OCH2CH3), 15.9 (m, CH3), 54.0 (s, OCH3),
59.3 (q, Cα, 2JCF = 30.2 Hz), 64.1 (s, OCH2), 123.3 (q, CF3, 1JCF = 281.0 Hz), 158.5
(m, CO), 164.3 (m, NHCO), 165.4 (m, COOCH3)
HRMS calculated for C9H12F3NO5 (M+) 271.0667, found 271.0668
- 105 -
Experimental section
Methyl 2-(diethylphosphonoformamido)-3,3,3-trifluoro-2-methylpropanoate (10)
Yield 58%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.42 (t, CH3, 6H, 3JHH = 6.8 Hz), 1.85 (s, CH3, 3H),
3.85 (s, OCH3, 3H), 4.22 (m, CH2, 4H), 7.81 (s, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -77.2 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -1.8 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.0 and 16.2 (d, CH3, 3JCP=6.0Hz), 16.9 (m, CH3),
54.7 (s, OCH3), 59.9 and 60.1 (both q, Cα, 2JCF = 28.8 Hz), 64.0 and 64.2 (d, OCH2),
121.8 and 122.0 (both q, CF3, 1JCF = 283.0 Hz), 163.5 (d, 1JCP = 122.0 Hz), 165.4
(CO)
HRMS calculated for C10H17F3NO6P (M+) 335.0745, found 335.0747
Methyl N-(ethoxyoxalyl)-3,3,3-trifluoro-2-phenylalaninate (11)
Yield 52%, white solid, mp 64-69oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.42 (t, 3H, CH3, 3JHH = 7.0 Hz), 3.93 (s, OCH3, 3H),
4.40 (q, OCH2, 2H, 3JHH = 7.0 Hz), 7.53 (s, HAR, 5H), 8.21 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -72.2 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 14.1 (s, CH3), 53.2 (s, OCH3), 64.0 (s, OCH2), 66.3
(q, Cα, 2JCF = 29.8 Hz), 122.2 (q, CF3, 1JCF = 285.0 Hz), 127.5, 128.3, 129.5, 136.6
(CHAR), 157.3 (m, CO), 164.3 (m, NHCO), 165.4 (m, COOCH3)
HRMS calculated for C14H14F3NO5 (M+) 333.0824, found 333.0826
- 106 -
Experimental section
Methyl N-[(diethylphosphonoformyl]-3,3,3-trifluoro-2-phenylalaninate (12)
Yield 49 %, pale yellow solid, mp 74-78oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.23 (m, CH3, 6H), 3.91 (s, OCH3, 3H), 4.30 (m,
OCH2, 4H), 7.61 (s, HAR, 5H), 7.92 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -72.1 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -1.6 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.1 and 16.3 (d, CH3, 3JCP=6.1Hz), 54.2 (OCH3),
62.0 and 62.3 (d, OCH2), 65.0 and 65.2, (both q, Cα, 2JCF = 28.2 Hz), 121.1 and
121.3 (both q, CF3, 1JCF = 281.0 Hz), 127.2, 128.1, 129.3, 133.7 (CHAR), 164.5 (d, 1JCP
= 121.3 Hz), 165.0 (CO)
HRMS calculated for C15H19F3NO6P (M+) 397.0902, found 397.0904
Methyl N-(ethoxyoxalyl)-(trifluoromethyl)-phenylalaninate (13)
Yield 59 %, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.36 (t, CH3, 3H, 3JHH = 7.2 Hz), 3.55 (d, 1H, JHH =
14.0 Hz), 3.90 (s, OCH3, 3H), 4.24 (d, 1H, JHH = 14.0 Hz), 4.33 (q, OCH2, 2H, 3JHH =
7.2 Hz), 7.22 (m, HAR, 4H), 7.92 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -73.4 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 14.0 (s, CH3), 35.9 (m, CH2), 53.8 (s, OCH3), 63.8
(s, OCH2), 65.1 (q, Cα, 2JCF = 28.5 Hz), 122.9 (q, CF3, 1JCF = 288.2 Hz), 125.4, 127.2,
131.4, 136.5 (CHAR), 155.1 (m, CO), 162.3 (m, NHCO), 166.2 (m, COOCH3)
HRMS calculated for C15H16F3NO5 (M+) 347.0980, found 347.0981
- 107 -
Experimental section
Methyl N-(diethylphosphonoformyl]-Į-(trifluoromethyl)phenylalaninate (14)
Yield 55%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.32 (m, CH3, 6H), 3.57 (d, 1H, JHH = 7.0 Hz), 3.92
(s, OCH3, 3H), 4.11 (m, 1H), 4.26 (m, OCH2, 4H), 7.13 (m, HAR, 4H), 7.71 (s, NH,
1H).
19
F NMR (188.31 MHz, CDCl3) δ: -73.4 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -1.3 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 15.9 and 16.1 (d, CH3, 3JCP=6.1Hz), 34.5 (m, CH2),
55.3 (OCH3), 61.7 and 62.0 (d, OCH2), 65.2 and 65.5, (both q, Cα, 2JCF = 29.1 Hz),
122.0 and 122.3 (both q, CF3, 1JCF = 284.1 Hz), 127.4, 129.5, 130.3, 134.9 (CHAR),
158.6 (CO), 162.7 (d, 1JCP = 120.3 Hz)
HRMS calculated for C16H21F3NO6P (M+) 411.1058, found 411.1059
Methyl 2-{ethoxyoxalylamino}-2-(trifluoromethyl)pent-4-enoate (15)
Yield 43%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.43 (t, CH3, 3H, 3JHH = 7.2 Hz), 2.97 (m, 1H), 3.63
(m, CH, 1H), 3.88 (s, OCH3, 3H), 4.36 (q, OCH2, 2H, 3JHH = 7.2 Hz), 4.99 (m, CH,
1H), 5.22 (m, CH2, 2H), 7.93 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -73.2 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 13.7 (s, CH3), 38.2 (m, CH2), 53.3 (s, OCH3), 62.9
(s, OCH2), 70.2 (q, Cα, 2JCF = 29.8 Hz), 121.7 (s, =CH2), 122.3 (q, CF3, 1JCF = 282.2
Hz), 132.4 (m, CH=CH2), 153.6 (m, CO), 160.3 (m, NHCO), 165.8 (m, COOCH3)
HRMS calculated for C11H14 F3NO5 (M+) 297.0824, found 297.0826
- 108 -
Experimental section
Methyl
2-{(diethylphosphonoformyl}amino}-2-(trifluoromethyl)pent-4-enoate
(16)
Yield 48 %, colorless oil
1
H NMR, (200.13 MHz, CDCl3) δ: 1.35 (t, CH3, 6H, 3JHH = 7.0 Hz), 2.98 (m, CH2, 2H),
3.51 (m, CH, 1H), 3.92 (s, OCH3, 3H), 4.23 (m, OCH2, 4H), 4.87 (m, CH, 1H), 5.19
(m, CH2, 2H), 7.75 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -73.2 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -1.4 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 15.8 and 16.0 (d, CH3, 3JCP=6.1Hz), 36.9 (m, CH2),
53.9 (s, OCH3), 62.5 and 62.7 (d, OCH2), 69.0 and 69.2, (both q, Cα, 2JCF = 29.7 Hz),
120.9 (=CH2), 122.5 and 122.7 (both q, CF3, 1JCF = 282.1 Hz), 131.5 (CH=CH2), 156.7
(CO), 162.7 (d, 1JCP = 120.9 Hz)
HRMS calculated for C12H19 F3NO6P (M+) 361.0902, found 361.0903
Methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(1H-indol-3-yl)alaninate (17)
A mixture of indol (8.0 mmol) and imine (7) (8.0 mmol) in anhydrous diethyl ether (10
ml) was stirred at r.t. overnight. The white precipitate was filtered off, washed with
ether to give analytically pure 17.
Yield 93%, white crystals, mp 178-180oC
1
H NMR, (200.13 MHz, CDCl3) δ: 1.45 (q, CH3, 3H, 3JHH = 6.9 Hz), 3.86 (s, OCH3,
3H), 4.44 (q, OCH2, 2H, 3JHH = 6.9 Hz), 7.33 (m, HAR, 4H), 7.84 (d, 1H, JHH = 7.8 Hz),
8.43 (s, 1H), 8.62 (s, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -72.2 (s, CF3)
- 109 -
Experimental section
13
C NMR (50.32 MHz, CDCl3) δ: 13.9 (s, CH3), 53.8 (s, OCH3), 64.1 (s, OCH2), 75.8
(q, Cα, 2JCF = 30.2 Hz), 112.2, 118.4, 118.9, 120.3, 120,4 (CHAR), 125.6 (q, CF3, 1JCF
= 280.1 Hz), 128.1, 129.3, 136.4 (CHAR), 155.6 (NHCO), 160.8 (COOCH3), 164.3
(CO)
HRMS calculated for C16H15F3N2O5 (M+) 372.0933, found 372.0934
Methyl
N-[(diethylphosphonoformyl]-3,3,3-trifluoro-2-(1H-indol-3-yl)alaninate
(18)
A mixture of indol (8.0 mmol) and imine (8) (8.0 mmol) in anhydrous diethyl ether (10
mL) was stirred at r.t. overnight. The light yellow precipitate was filtered off, washed
with ether to give analytically pure 17.
Yield 85%, light yellow solid, mp 108-110 oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.31 (m, CH3, 6H), 3.82 (s, OCH3, 3H), 4.21 (m,
OCH2, 4H), 7.43 (m, HAR, 4H), 7.63 (d, 1H, JHH = 7.8 Hz), 8.23 (s, 1H), 8.90 (s, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -72.2 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -1.2 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.6 and 16.7 (d, CH3, 3JCP=6.0Hz), 54.5 (OCH3),
65.4 and 65.5 (d, OCH2), 65.7 (q, Cα, 2JCF = 28.3 Hz), 105.3 (m, C-CĮ), 112.5, 119.2,
121.2, 123.2, 124.6 (CHAR), 124.9 (q, CF3, 1JCF = 280.1 Hz), 127.0, 129.3, 136.4
(CHAR), 165.6 (d, 1JCP = 122.3 Hz), 168.2 (CO)
HRMS calculated for C17H20F3N2O6P (M+) 436.1011, found 436.1010.
- 110 -
Experimental section
General procedure for the preparation of indols 19 and 20
A mixture of 2-methylindol (8.0 mmol) and appropriate imine (7, 8) (8.0 mmol) in
anhydrous CHCl3 was heated at 60-70oC for 6-8 hours. A solvent was removed
under reduced pressure; the product was isolated by flash chromatography on silica
gel (eluent: ethyl acetate/hexanes).
Methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(2-methyl-1H-indol-3-yl)alaninate (19)
Yield 62 %, white solid, mp 107-109 oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.43 (t, CH3, 3H, 3JHH= 7.1 Hz), 2.46 (s, CH3, 3H),
3.92 (s, OCH3, 3H), 4.42 (q, OCH2, 2H, 3JHH = 7.1 Hz), 7.26 (m, 2H), 7.38 (m, 1H),
7.65 (br.s, 1H), 8.71 (s, 1H), 8.80 (s, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -71.2 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 11.9 (s, CH3), 13.8 (s, OCH2CH3), 54.8 (s, OCH3),
64.5 (s, OCH2), 65.1 (q, Cα, 2JCF = 28.2 Hz), 111.8 (m, C-CĮ), 113.9, 118.5, 120.1,
121.1 (CHAR), 125.6 (q, CF3, 1JCF = 281.1 Hz), 128.6, 136.2, 141.4 (CHAR), 158.5
(CO), 164.8 (NHCO), 167.3 (COOCH3)
HRMS calculated for C17H17F3N2O5 (M+) 386.1089, found 386.1090
Methyl
N-(diethylphosphonoformyl)-3,3,3-trifluoro-2-(2-methyl-1H-indol-3-
yl)alaninate (20)
Yield 50 %, pale yellow oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.39 (m, CH3, 6H), 2.48 (s, CH3, 3H), 3.92 (s, OCH3,
3H), 4.26 (m, OCH2, 4H), 7.18 (m, 2H), 7.21 (m, 1H), 7.35 (br.s, 1H), 8.55 (s, 1H),
8.69 (s, 1H)
- 111 -
Experimental section
19
F NMR (188.31 MHz, CDCl3) δ: -71.8 (s, CF3).
31
P NMR (80.99 MHz, CDCl3) δ: -2.4 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 13.4 (s, CH3), 16.2 and 16.4 (d, CH3, 3JCP=6.1Hz) ,
53.9 (s, OCH3), 62.4 and 62.6 (d, OCH2), 64.5 and 64.6 (both q, Cα, 2JCF = 30.8 Hz),
106.2 (m, C-CĮ), 113.5, 117.4, 121.2, 122.8 (CHAR), 126.6 and 127.0 (q, CF3, 1JCF =
283.0 Hz), 135.4 (CHAR), 145.5 (C-CH3), 165.2 (d, 1JCP = 123.5 Hz), 167.2 (CO)
HRMS calculated for C18H22F3N2O6P (M+) 450.1167, found 450.1168.
General procedure for the preparation of furans and pyrroles (21- 24)
To a 0oC solution of the corresponding furan or pyrrole (8.0 mmol) in ether (10 mL) a
solution of appropriate imine (7, 8) (4.0 mmol) in 5 ml of ether was added. The
mixture was allowed to warm up to r.t. and was stirred until
19
F NMR spectrum
indicated the full conversion of imine. The solvent was removed under reduced
pressure. The crude residue was purified by flash chromatography eluting with
AcOEt/hexanes.
Methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(2-furyl)alaninate (21)
Yield 39 %, white solid, mp 74-79 oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.47 (t, CH3, 3H, 3JHH = 7.2 Hz), 3.83 (s, OCH3, 3H),
4.45 (q, OCH2, 2H, 3JHH = 7.2 Hz), 6.43 (m, HAR, 1H, JHH = 2.8 Hz), 6.62 (d, HAR, 1H,
JHH = 3.2 Hz), 7.43 (m, HAR, 1H), 8.22 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -73.3 (s, CF3)1
- 112 -
Experimental section
13
C NMR (50.32 MHz, CDCl3) δ: 14.2 (s, CH3), 53.9 (s, OCH3), 63.5 (s, OCH2), 64.0
(q, Cα, 2JCF = 29.0 Hz), 104.8, 109.7 (CHAR), 121.9, (q, CF3, 1JCF = 278.1 Hz), 139.8
(CHAR), 151.5 (m, C- Cα), 157.8 (s, CO), 165.9 (m, NHCO), 166.3 (m, COOCH3)
HRMS calculated for C12H12F3NO6 (M+) 323.0617, found 323.0619
Methyl N-(diethylphosphonoformyl]-3,3,3-trifluoro-2-(2-furyl)alaninate (22)
Yield 40 %, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.43 (t, CH3, 6H, 3JHH = 7.1 Hz), 3.88 (s, OCH3, 3H),
4.25 (m, OCH2, 4H), 6.42 (m, HAR, 1H), 6.63 (d, HAR, 1H, JHH = 3.4 Hz), 7.42 (m, HAR,
1H), 8.07 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -73.3 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -1.8 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.1 and 16.3 (d, CH3, 3JCP=5.9Hz), 54.3 (s, OCH3),
62.2 and 62.4 (d, OCH2), 65.7 and 65.9 (both q, Cα, 2JCF = 31.3 Hz), 100.8, 108.5,
121.8 (CHAR) 122.1 (both q, CF3, 1JCF = 280.0 Hz), 140.4 (m, C- Cα), 143.2 (CO),
164.7 (d, 1JCP = 122.5 Hz), 166.1 (COOCH3)
HRMS calculated for C13H17F3NO7P (M+) 387.0695, found 387.0694
Methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(1-methyl-1H-pyrrol-2-yl)alaninate (23)
Yield 38 %, white solid, mp 95-97 oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.42 (t, CH3, 3H, 3JHH= 6.8 Hz), 3.75 (s, CH3, 3H),
3.82 (s, OCH3, 3H), 4.46 (q, OCH2, 2H, 3JHH = 6.8 Hz), 6.25 (s, HAR, 1H), 6.64 (d,
HAR, 1H, JHH = 2.4 Hz), 7.63 (s, HAR, 1H), 8.21 (s, NH, 1H)
- 113 -
Experimental section
19
F NMR (188.31 MHz, CDCl3) δ: -73.3 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 14.3 (s, CH3), 36.9 (N-CH3), 53.8 (s, OCH3), 64.2 (s,
OCH2), 67.5 (q, Cα, 2JCF = 34.0 Hz), 107.5, 114.6, 119.8, 121.5 (CHAR), 123.5 (q,
CF3, 1JCF = 286.0 Hz), 155.6 (m, C- Cα), 160.3 (m, CO), 165.7 (m, COOCH3)
HRMS calculated for C13H15F3N2O5 (M+) 336.0933, found 336.0934
Methyl
Į-(diethylphosphonoformamido)-Į-(trifluoromethyl)-1H-pyrrole-3-
acetate (24)
Yield 42%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.36 (t, CH3, 6H, 3JHH= 7.2 Hz), 3.65 (s, CH3, 3H),
3.82 (s, OCH3, 3H), 4.33 (m, OCH2, 4H), 6.24 (s, HAR, 1H), 6.63 (d, HAR, 1H, JHH= 2.6
Hz), 7.54 (s, HAR, 1H), 8.52 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -73.3 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -1.1 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.3 and 16.4 (d, CH3, 3JCP=6.0Hz), 40.1 (N-CH3),
53.8 (OCH3), 61.2 and 61.4 (d, OCH2), 62.0 and 62.2 (both q, Cα, 2JCF = 28.1 Hz),
104.4, 105.2, 120.3 (CHAR), 123.5 (q, CF3, 1JCF = 279.7 Hz), 135.2 (m, C- Cα), 158.6
(m, COOCH3), 165.8 (d, 1JCP = 122.5 Hz)
HRMS calculated for C14H20F3N2O6P (M+) 400.1011, found 400.101
- 114 -
Experimental section
Methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(5-methyl-3-oxo-2-phenyl-2,3-dihydro1H-pyrazol-4-yl)-alaninate (25)
A mixture of 1-phenyl-4-methylpyrazol-2-on (6.0 moll) and imine 7 (6.0 mmol) in
anhydrous diethyl ether (10 ml) was stirred at r.t. overnight. The white precipitate was
filtered off, washed with ether to give analytically pure 25
Yield 76%, white solid, mp 84-88 oC
1
H NMR (200.13 MHz, DMSO) δ: 1.41 (t, CH3, 3H, 3JHH = 7.2 Hz), 2.24 (s, CH3, 3H),
3.78 (s, OCH3, 3H), 4.35 (q, OCH2, 2H, 3JHH = 7.2 Hz), 7.37-7.51 (m, HAR, 3H), 7.62
(m, HAR, 2H), 12.10 (s, NH, 1H)
19
F NMR (188.31 MHz, DMSO) δ: -76.3 (s, CF3)
13
C NMR (50.32 MHz, DMSO) δ: 11.7 (s, CH3), 14.6 (s, OCH2CH3), 54.2 (s, OCH3),
62.8 (q, Cα, 2JCF = 30.7 Hz), 64.2 (s, OCH2), 119,5 (m, Cα-C=), 121.5 (CHAR), 122.6
(q, CF3, 1JCF =272.0 Hz), 127.3, 135.3 (CHAR), 140.5 (s, N-C=), 156.2 (m, NHCO),
158.3 (m, =C-CH3), 159.6 (s, COCO), 161.2 (q, COOCH3), 165.2 (m, CO)
HRMS calculated for C18H18F3N3O6 (M+) 429.1148, found 429.1147
Methyl
N-(diethylphosphonoformyl)-3,3,3-trifluoro-2-(5-methyl-3-oxo-2-phenyl-
2,3-dihydro-1H-pyrazol-4-yl)alaninate (26)
A mixture of 1-phenyl-4-methylpyrazol-2-on (6.0 mol) and imine 7 (6.0 mmol) in
anhydrous diethyl ether (10 mL) was stirred at r.t. overnight. The white precipitate
was filtered off, washed with ether to give analytically pure 26.
Yield 55%, pale yellow solid, mp 164-168 oC
1
H NMR (200.13 MHz, DMSO) δ: 1.37 (t, CH3, 6H, 3JHH = 7.0 Hz), 1.91 (s, CH3, 3H),
3.63 (s, OCH3, 3H), 4.15 (m, OCH2, 4H), 7.19-7.30 (m, HAR, 3H), 7.92 (m, HAR, 2H),
13.11 (s, NH, 1H)
- 115 -
Experimental section
19
F NMR (188.31 MHz, DMSO) δ: -76.3 (s, CF3)
31
P NMR (80.99 MHz, DMSO) δ: -0.7 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, DMSO) δ: 12.2 (s, CH3), 16.3 and 16.5 (d, CH3, 3JCP=6.1 Hz),
53.3 (s, OCH3), 64.4 and 64.6 (d, OCH2), 67.8 (q, Cα, 2JCF = 29.3 Hz), 107.0 (m, CαC=), 121.4 (CHAR), 124.8 (q, CF3, 1JCF = 280.0 Hz), 125.1, 129.3, 139.5 (CHAR), 160.7
(=C-CH3), 162.3 (m, COOCH3), 163.5 (m, CO), 164.7 (d, 1JCP = 122.5 Hz)
HRMS calculated for C19H23F3N3O7P (M+) 493.1226, found 493.1225
Methyl 2-[4-(dimethylamino)phenyl]-N-(ethoxyoxalyl]3,3,3-trifluoroalaninate (27)
To a chilled (-40 oC) solution of N,N-dimethylaniline (8.0 mmol) in ether (10 mL) a
solution of imine 6 (8.0 mmol) in 5 mL of ether was added. The mixture was allowed
to warm to r.t. and stirred until the
19
F NMR spectrum indicated the full conversion of
the imine. A solvent was removed under reduced pressure. The crude residue was
purified by flash chromatography on silica eluting with AcOEt/hexanes.
Yield 46%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.44 (t, CH3, 3H, 3JHH = 7.0 Hz), 2.95 (s, CH3, 6H),
4.03 (s, OCH3, 3H), 4.2 (q, OCH2, 2H, 3JHH = 7.0 Hz), 6.85 (d, HAR, 2H, JHH = 9.0 Hz),
7.32 (d, HAR, 2H, JHH = 9.0 Hz), 7.80 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -71.3 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 14.1 (s, CH3), 40.4 (s, N-CH3), 55.8 (s, OCH3), 63.6
(s, OCH2), 70.5 (q, Cα, 2JCF = 31.0 Hz), 113.5, 121.5, 121.6 (CHAR), 123.4 (q, CF3,
1
JCF =281.0 Hz), 125.6, 128.8, 128.9 (CHAR), 149.5 (s, C-NCH3), 160.1 (m, NHCO),
161.3 (s, COCO), 165.7 (m, COOCH3)
HRMS calculated for C16H19F3N2O5 (M+) 376.1246, found 376.1247
- 116 -
Experimental section
Methyl
N-(diethylphosphonoformyl)-2-[4-(dimethylamino)phenyl]-3,3,3-
trifluoroalaninate (28)
To a chilled (-40 oC) solution of N,N-dimethylaniline (8.0 mmol) in ether (10 mL) a
solution of imine 7 (8.0 mmol) in 5 ml of ether was added. The mixture was allowed
to warm to r.t. and stirred until the
19
F NMR spectrum indicated the full conversion of
the imine. A solvent was removed under reduced pressure. The crude residue was
purified by flash chromatography on silica eluting with AcOEt/hexanes.
Yield 43%, yellow oil
1
H NMR (200.13 MHz, CD3CN) δ: 1.33 (t, CH3, 6H, 3JHH = 7.2 Hz), 2.91 (s, CH3, 6H),
3.76 (s, OCH3, 3H), 4.13 (m, OCH2, 4H), 6.78 (d, HAR, 2H, JHH = 9.0 Hz), 7.34 (d, HAR,
2H, JHH = 9.0 Hz), 7.93 (s, NH, 1H)
19
F NMR (188.31 MHz, CD3CN) δ: -71.3 (s, CF3)
31
P NMR (80.99 MHz, CD3CN) δ: 3.8 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCN3) δ: 16.1 and 16.3 (d, CH3, 3JCP=6.0 Hz), 39.9 (s, N-
CH3), 53.5 (s, OCH3), 61.2 and 61.3 (d, OCH2), 64.8 and 64.9 (both q, Cα, 2JCF =
28.3 Hz), 111.4, 120.3 (CHAR), 122.9 and 123.0 (both q, CF3, 1JCF = 285.0 Hz), 127.1
(CHAR), 150.7 (s, C-NCH3), 161.5 (m, COOCH3), 163.4 (d, 1JCP = 124.1 Hz)
HRMS calculated for C17H24F3N2O6P (M+) 440.1324, found 440.1325
General procedure for the reactions of 7 and 8 with NaBH4
To a 0 oC solution of imine 2 (8.0 mmol) in ether (10 ml) a sodium borohydride (6.0
mmol, powder from Aldrich) was carefully added. The resulting suspension was
stirred overnight at room temperature under nitrogen. The reaction mixture was
quenched with 1N HCl and extracted with ether (2 x 50 mL). The combined organic
layers were washed with brine (25 mL), dried over MgSO4 and filtered. The solvent
- 117 -
Experimental section
was removed under reduced pressure and the crude product was purified by flash
chromatography on silica gel (eluent: ethyl acetate/hexanes).
Methyl N-[ethoxyoxalyl]-3,3,3-trifluoroalaninate (29)
Yield 69 %, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.36 (t, CH3, 3H, 3JHH = 7.2 Hz), 3.79 (s, OCH3, 3H),
4.35 (m, OCH2, 2H), 6.15 (m, 1H), 8.23 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -72.0 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 14.0 (s, CH3), 53.5 (s, OCH3), 59.9 (q, Cα, 2JCF =
32.0 Hz), 64.6 (s, OCH2), 125.4 (q, CF3, 1JCF =288.2 Hz), 159.1 (s, COCO), 161.4 (m,
COOCH3), 167.7 (m, NHCO)
HRMS calculated for C8H10F3NO5 (M+) 257.0511, found 257.0512
Methyl 3-(diethylphosphonyl)-2-(trifluoromethyl)-2H-azirine-2-carboxylate (30)
Yield 57%, colorless oil.
1
H NMR (200.13 MHz, CDCl3) δ: 1.40 (m, CH3, 6H), 4.12 (s, OCH3, 3H), 4.34 (m,
OCH2, 4H)
19
F NMR (188.31 MHz, CDCl3) δ: -62.7 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -3.2 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.4 and 16.5 (d, CH3, 3JCP=6.0 Hz), 60.4 (s, OCH3),
64.9 and 64.8 (d, OCH2), 105.9 and 105.8 (both q, 2JCF = 40.9 Hz), 120.5 and 120.6
(both q, CF3, 1JCF =267.0 Hz), 146.5 (d, 1JCP = 278.3 Hz), 158.9 (m, COOCH3)
IR (thin layer) ν/cm-1: 1025, 1046 (P-O-C, C-O-C), 1271 (P=O), 1652 (C=O), 1765
(C=N)
- 118 -
Experimental section
HRMS calculated for C9H13F3NO5P (M+) 303.0483, found 303.0482
Methyl 3-(diethylphosphonyl)-2-(trifluoromethyl)-2H-aziridine-2-carboxylate (31)
Yield 62%, light yellow oil.
1
H NMR (200.13 MHz, CDCl3) δ: 1.38 (m, CH3, 6H), 2.55 (b. s. NH, 1H), 3.23 (dd,
CH-PO, 1H), 4.12 (s, OCH3, 3H), 4.32 (m, OCH2, 4H)
19
F NMR (188.31 MHz, CDCl3) δ: -62.5 (s, CF3)
31
P NMR (80.99 MHz, CDCl3) δ: -3.1 (m, P(O)(OEt)2)
Methyl N-(oxalyl)-(trifluoromethyl)-phenylalaninate acid (32)
1eq of Methyl N-(ethoxyoxalyl)-(trifluoromethyl)-phenylalaninate was dissolved in
mixture of methanol and water (4:1). Then, 4eq of KHCO3 was added and the
reaction mixture was stirred at room temperature 24 h. Solvents were evaporated
and 10 mL of water was added. The reaction mixture was quenched with 1N HCl
containing NaCl and extracted with ethyl acetate (3 x 30 mL). The combined organic
layers were dried over Na2SO4 and filtered. The solvent was removed under reduced
pressure and the product was recrystallized from the mixture ethyl acetate/ hexane.
Yield 89 %, white crystals mp 78-81oC
1
H NMR (200.13 MHz, CDCl3) δ: 3.56 (d, 1H, JHH = 14.2 Hz), 3.95 (s, OCH3, 3H), 4.17
(d, 1H, JHH = 14.3 Hz), 7.29 (m, HAR, 4H), 8.22 (s, NH, 1H)
19
F NMR (188.31 MHz, CDCl3) δ: -73.4 (s, CF3)
13
C NMR (50.32 MHz, CDCl3) δ: 33.4 (m, CH2), 54.8 (s, OCH3), 68.7 (q, Cα, 2JCF =
28.7 Hz), 123.2 (q, CF3, 1JCF = 288.7 Hz), 125.2, 127.9, 133.1, 135.5 (CHAR), 155.5
(m, CO), 161.2 (m, NHCO), 163.1 (m, COOCH3)
- 119 -
Experimental section
Methyl
N-oxalyl-3,3,3-trifluoro-2-(5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-alaninate acid (33)
1eq of methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(5-methyl-3-oxo-2-phenyl-2,3-dihydro1H-pyrazol-4-yl)-alaninate was dissolved in mixture of methanol and water (4:1).
Then, 4eq of KHCO3 was added and the reaction mixture was stirred at room
temperature 24 h. Solvents were evaporated and 10 mL of water was added. The
reaction mixture was quenched with 1N HCl containing NaCl and extracted with ethyl
acetate (3 x 30 mL). The combined organic layers were dried over Na2SO4 and
filtered. The solvent was removed under reduced pressure and the product was
recrystallized from the mixture ethyl acetate/ hexane
Yield 87%, white solid, mp 90-95 oC
1
H NMR (200.13 MHz, DMSO) δ: 2.24 (s, CH3, 3H), 3.74 (s, OCH3, 3H), 7.30 (t, HAR,
1H, 3JHH = 7.2 Hz), 7.49 (t, HAR, 2H, 3JHH = 7.6 Hz), 7.67 (d, HAR, 2H, 2JHH = 12.01 Hz),
11.91 (s, NH, 1H)
19
F NMR (188.31 MHz, DMSO) δ: -76.36 (s, CF3)
13
C NMR (50.32 MHz, DMSO) δ: 11.69 (s, CH3), 53.99 (s, OCH3), 62.72 (q, Cα, 2JCF
= 30.6 Hz), 119.50 (m, Cα-C=), 121.38 (CHAR), 123.60 (q, CF3, 1JCF =272.0 Hz),
127.21, 135.98 (CHAR), 140.51 (s, N-C=), 156.24 (m, NHCO), 158.32 (m, =C-CH3),
157.67 (s, COCO), 161.42 (q, COOCH3), 165.82 (m, CO)
MS (EI, 70eV, 200oC) m/z %; 401 (18) [M]+, 312 (100) [M-88]+, 207 (21) [M-357]+, 77
(37) [M-314]+, 59 (2) [M-342]+, 44 (4) [M-357]+
- 120 -
Experimental section
Tetraethyl ethylidenebis (phosphonate) (36)
was obtained via the references (155, 156)
ƒ
ȕ- Methoxyethylene diphosphonate (35)
Paraformaldehyde (10.0 g; 0.33 mol) and diethylamine (5.2g; 0.06 mol) were
combined with 200 mL of dry methanol and the mixture was warmed up until clear.
The heat was removed and tetraethyl methylenebis (phosphonate) (20.0 g; 0.06 mol)
was added. The mixture was refluxed for the next 24h. After this period of time, an
additional 200 mL of methanol was added. The solution was concentrated under
vacuum at 35oC. Then, 100 mL of dry toluene was added and the solution was once
again concentrated. This step was repeated twice in order to ensure complete
removal of methanol from the product
Yield 89 %, orange oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.25 (t, CH3, 12H, 3JHH = 7.0 Hz), 2.48 (tt, PCHP,
1H, 2JPH = 24.0 Hz, 3JH = 7.0 Hz), 3.45 (s, OCH3, 3H), 3.71 (td, CH2, 2H, 2JPH= 16.5
Hz, 3JHH= 5.0 Hz), 3.89 (m, OCH2, 8H)
31
P NMR (80.99 MHz, CDCl3) δ: 21.2 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.71 (d, CH3, 3JCP = 7.2 Hz), 39.40 (t, PCP, 3JCP =
7.0 Hz), 58.72 (s, OCH3), 63.02 (d, OCH2, 2JCP= 5.1 Hz), 68.1 (t, CH2, 2JCP= 4.8 Hz)
ƒ
Tetraethyl ethylidene diphosphonate (36)
ȕ- methoxyethylene 1, 1- bis (phosphonate) (15.0 g; 0.05 mol) was dissolved in 150
mL of dry toluene was p-toluenesulfonic acid monohydrate (0.07 g) was added. The
mixture was refluxed using a Dean- Stark trap in order to remove the rest of the
methanol. After 10h solution was concentrated. The crude product was diluted in 200
mL of chloroform was washed with water (3 x 100 mL). The organic layer was dried
- 121 -
Experimental section
over MgSO4 and filtered. The solvent was removed under reduced pressure and the
crude product was purified. An analytical pure sample was obtained by flash
chromatography on silica gel (eluent: acethone/hexane).
Yield 83%, light yellow oil
1
H NMR (200.13MHz, CDCl3) δ: 1.33 (t, CH3, 12H, 3JHH = 7.0 Hz), 4.15 (m, OCH2,
8H), 6.98 (dd, CH2, 2H, trans J3PH = 40 Hz, cis J3PH = 36 Hz)
31
P NMR (80.99 MHz, CDCl3) δ: 14.43 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.21 (d, CH3, 3JCP = 3.5 Hz), 62.70 (d, OCH2, 2JCP=
3.8 Hz), 148.10 (s, =CH2)
IR (thin layer) ν/cm-1: 1028 (P-O-C), 1241 (P=O)
Tetraethyl but-3-yne-1,1-diyldiphosphonate (37)
To solution of ethylidenbisphosphonate 1 (10 g, 34.4 mmol) in dry THF (100 mL) a
slurry of sodium acethylenide in xylenes (9.1 mL, 18 % solution) was added dropwise
at -15 oC. Reaction mixture was allowed to warm to r.t. and stirred overnight. To
reaction solution ether (100 mL) and 1N HCl (50 mL) were added. An organic layer
was washed with 1N HCl (50 mL), brine (2 x 50 mL) and dried over magnesium
sulfate. After evaporation of the solvent under reduced pressure the product was
used for the further reactions without purification.
Yield 92%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.31 (t, 12H, CH3, 3JHH = 7.2 Hz), 2.03 (s, 1H, C≡H),
2.61-2.92 (m, 3H, CHP+CH2), 4.22-4.28 (m, 8H, OCH2)
31
P NMR (80.99 MHz, CDCl3) δ: 22.8 (m, P(O)(OEt)2)
- 122 -
Experimental section
13
C NMR (50.32 MHz, CDCl3) δ: 16.7 (d, CH3, 3JCP = 6.1 Hz), 39.6 (CH, t, 1JCP =
134.3), 63.3 (d, OCH2, 2JCP = 6.5 Hz), 70.4 (HC≡), 81.6 (t, CH2C≡, 3JCP = 9.4)
IR (thin layer) ν/cm-1: 1025 (P-O-C), 1249 (P=O), 2120 (C≡C)
HRMS calculated for C12H26O6P2 (M+) 326.1048, found 326.1040
Tetraethyl hepta-1,6-diyne-4,4-diyldiphosphonate (38)
was obtained via the reference (157)
Methylenebisphosphonate (5g, 17.4mmol) was added dropwise to dry THF (75 mL)
containing sodium hydride (0.8g, 34.7mmol). Reaction mixture was cooled down till
0oC. Then, propargyl bromide (4.2g, 34.7mmol) was slowly added dropwise to the
reaction mixture and stirred at room temperature for 3h and then 4h at 40 oC. To
reaction solution water was added in order to quench reaction. A water phase was
extracted with ether and then organic phase was dried over MgSO4. Product was
purified by flash column chromatography.
Yield 78% (white crystals) m.p. 57- 61oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.34 (t, 12H, CH3, 3JHH =7.0), 2.04 (s, 2H, C≡H),
2.90 (td, 4H, CH2, 3JHH = 3.1, 2JHP = 15.9 Hz), 4.23 (quintet, 8H, OCH2)
31
P NMR (80.99 MHz, CDCl3) δ: 24.3 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 14.3 (d, CH3, 3JCP = 6.1 Hz), 19.0 (CH2), 41.7 (t,
C(CH2)2, 1JCP = 133.8 Hz), 61.1 (d, OCH2, 2JCP = 7.2 Hz), 69.6 (HC≡), 76.8 (t, CH2C≡,
3
JCP = 10.9 Hz)
IR (thin layer) ν/cm-1: 1025 (P-O-C), 1255 (P=O), 2120 (C≡C)
- 123 -
Experimental section
HRMS calculated for C15H26O6P2 (M+) 364.1205, found 364.1204
Typical procedure for the synthesis of aryl azides
was obtained via the reference (163)
Aryl bromide (2 mmol), NaN3 (4 mmol), sodium ascorbate (0.1 mmol), CuI (0.2
mmol), ligand (N,N-dimethylethane-1,2-diamine) (0.3 mmol), and 4 mL of mixture
EtOH: H2O (7:3) were introduced into a round bottom flask equipped with condenser.
The reaction mixture was stirred under reflux at the atmosphere of nitrogen. The
progress of the reaction was controlled by TLC. When the conversion of the aryl
bromide into corresponding aryl azide was completed, the reaction mixture was
allowed to cool down to r.t. After filtration, the crude product was purified by flash
column chromatography giving the desired aryl azide.
1-azidobenzene (39)
Yield 88%, pale yellow oil, B.p. 49-50 oC, 5 mm Hg
1
H NMR (200.13 MHz, CDCl3) δ: 7.42-7.50 (m, 5H, HAr)
13
C NMR (50.32 MHz, CDCl3) δ: 128.5, 129.1 and 130.2 (CHAr), 136.4 (CArN)
IR (thin layer) ν/cm-1: 1560 (C=C), 2215 (N3), 3033 (C-H)
1-(azidomethyl)benzene (40)
Yield 85%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 5.53 (s, 2H, CH2), 7.28- 7-31 (m, 5H, HAR)
- 124 -
Experimental section
13
C NMR (50.32 MHz, CDCl3) δ: 55.3 (NCH2), 128.5, 129.3 (CHAr), 135.4 (CArN)
IR (thin layer) ν/cm-1: 1545 (C=C), 2218 (N3), 3035 (C-H)
8-azido-1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorooctane (41)
was obtained via reference (164)
To a round bottom flask, C6F13C2H4Br (0.1 mol), NaN3 (0.15 mol), 18-C-6 (0.5g) and
DMSO 100 mL were added successively. Then the mixture was stirred at 110oC. The
progress of the reaction was monitored by 19F NMR. When the peak belonging to
starting material dissapeared, the reaction mixture was cooled down till 50oC and
poured into ice water. Water layer was extracted with ether. Combined organic layers
were washed with saturated brine was dried over MgSO4. After removal of the
solvent, the product was purified by distillation.
Yield 92%, colorless oil, B.p. 92 oC, 45 mm Hg
1
H NMR (200.13 MHz, CDCl3) δ: 2.40 (m, 2H, CH2CF2, 3JHH = 7.2 Hz, 3JHF = 11.1 Hz),
3.61 (t, 2H, CH2N3, 3JHH = 7.0 Hz)
19
F NMR (188.31 MHz, CDCl3) δ: -82.0 (tt, 3F, CF3), -114.3 (m, 2F, CF2CH2), -123.0
(m, 2F, CF2CF3), -124.0 (m, 2F, CH2CF2CF2CF2), -124.6 (m, 2F, CH2CF2CF2), -127.2
(m, 2F, CF2CF2CF3)
IR (thin layer) ν/cm-1: 1518 (C=C), 2240 (N3)
Typical procedure for the synthesis of triazols
Method A
A mixture of organic azide (1.0 mmol), acetylene 2 (1.0 mmol), DIPEA (2.0 mmol)
and CuI (0.1 mmol) in THF (10 mL) was stirred at r.t. for 6÷8 h. The resulted reaction
- 125 -
Experimental section
mixture was treated with 1N HCl (15 mL), and extracted with ether (3 x 15 mL).
Combined organic layers were dried over MgSO4 and filtered. The solvent was
removed under reduced pressure and the crude product was purified by flash
chromatography on silica gel (acetone/petroleum ether).
Method B
Organic azide (2.0 mmol) and acetylene 2 (2.0 mmol) were suspended in 1:4 H2O-tBuOH (8 mL). To this was added CuSO4Â5H2O (5 M solution, 0.1 mmol, 5 mol%) and
sodium ascorbate (0.6 mmol). The mixture was stirred at r.t. for 24 h, at which time
TLC (silica, petroleum ether-acetone) indicated complete conversion. The resulted
solution was concentrated under reduced pressure (rotar evaporator). The residue
was dissolved in 30 mL of brine and then extracted with ethyl acetate (3 x 30 mL).
Combined organic layers were washed with 5 % aq NH4OH (2 x 10 mL), dried over
MgSO4, filtered and solvent was removed under vacuum to give analytically pure
product.
Tetraethyl 2-(1-phenyl-1H-1,2,3-triazol-4-yl)ethane-1,1-diyldiphosphonate (45)
Yield 87%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.31 (t, 12H, CH3, 3JHH = 7.1 Hz), 3.03 (tt, 1H, CHP,
3
JHH = 6.4, 2JHP = 23.1 Hz), 3.40 (dt, 2H, CH2, 3JHH = 6.4, 3JHP = 16.0 Hz), 4.22-4.31
(m, 8H, OCH2), 7.45-7.55 (m, 5H, HAR), 7.71 (s, 1H, CH)
31
P NMR (80.99 MHz, CDCl3) δ: 23.7 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.7 (d, CH3, 3JCP = 3.0 Hz), 16.8 (d, CH3, 3JCP = 2.5
Hz), 22.6 (CH2), 37.1 (t, CP, 1JCP = 132.9 Hz), 62.9 (OCH2), 63.3 (OCH2), 120.8
(ɋɇ=), 128.6, 128.9, and 130.2 (CHAr), 137.6 (CArN), 146.9 (ɋ=)
IR (thin layer) ν/cm-1: 1023 (P-O-C), 1255 (P=O), 1597 (C=C), 1500 (N=N)
- 126 -
Experimental section
HRMS calculated for C18H29N3O6P2 (M+) 445.1532, found 445.1543
Tetraethyl 2-(1-benzyl-1H-1,2,3-triazol-4-yl)ethane-1,1-diyldipshopshonate (46)
Yield 89%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.30 (t, 12H, CH3, 3JHH = 6.5Hz), 2.91 (tt, 1H, CHP,
3
JHH = 6.3, 2JHP = 23.0 Hz), 3.32 (dt, 2H, CH2, 3JHH = 6.4, 3JHP = 15.9 Hz), 4.21-4.26
(m, 8H, OCH2), 5.52 (s, 2H, CH2), 7.30-7.33 (m, 5HAr), 7.52 (s, 1H, CH)
31
P NMR (80.99 MHz, CDCl3) δ: 23.6 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.7 (CH3), 22.6, 37.1 (t, CP, 1JCP = 132.9 Hz), 55.2
(NCH2), 62.9 (d, OCH2, 2JCP = 6.5 Hz), 63.2 (d, OCH2, 2JCP = 6.2 Hz), 122.6 (ɋɇ=),
128.5 and 128.6 and 129.4 (CAr), 135.3 (CAr), 145.9 (C=)
IR (thin layer) ν/cm-1: 1024 (P-O-C), 1248 (P=O), 1498 (N=N), 1555 (C=C)
HRMS calculated for C19H31N3O6P2 (M+) 459.1688, found 459.1685
Tetraethyl
2-[1-(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)-1H-1,2,3-triazol-4-
yl]ethane-1,1-diyldiphosphonate (47)
Yield 92%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.31 (t, 12H, CH3, 3JHH = 7.0 Hz), 2.41-2.93 (m, 3H,
CHP+CH2), 3.40 (dt, 2H, CH2, 3JHH =6.1 Hz , 3JHP = 16.1 Hz), 4.22-4.31 (m, 8H,
OCH2), 4.72 (t, 2H, CH2, 3JHH = 7.2 Hz ), 7.61 (s, 1H, CH)
- 127 -
Experimental section
19
F NMR (188.31 MHz, CDCl3) δ: -81.9 (m, 3F, CF3), -114.3 (m, 2F, CF2CH2), -123.0
(m, 2F, CF2CF3), -124.0 (m, 2F, CH2CF2CF2CF2), -124.6 (m, 2F, CH2CF2CF2), -127.2
(m, 2F, CF2CF2CF3)
31
P NMR (80.99 MHz, CDCl3) δ: 23.7 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.6 (d, CH3, 3JCP = 3.0 Hz), 16.8 (d, CH3, 3JCP = 2.5
Hz), 22.5, 32.2 (t, CH2CF2, 2JCF = 21.7 Hz), 37.0 (t, CP, 1JCP = 133.1 Hz), 42.5
(NCH2), 62.9 (d, OCH2, 2JCP =6.5 Hz), 63.3 (d, OCH2, 2JCP =6.2 Hz), 109.3,
121.1(ɋɇ=), 123.4, 145.9 (C=)
IR (thin layer) ν/cm-1: 1028 (P-O-C), 1241 (P=O), 1367 and 1394 (CH2), 1550 (C=C),
3460 (N-H)
HRMS calculated for C20H28N3O6P2F13 (M+) 715.1245, found 715.1235
Tert-butyl{4-[2,2-bis(diethoxyphosphoryl)ethyl]-1H-1,2,3-triazol-1-yl}acetate (48)
Yield 64 %, colorless oil
1
H NMR (300.13 MHz, CDCl3) δ: 1.23 (s, 9H, ɋɇ3), 1.34 (t, 12H, ɋɇ3, 3JH-H = 7.08
Hz), 2.97-3.04 (m, 1H, ɋɇ), 3.38 (dt, 2H, CH2, 3JH-H =16.20 Hz), 4.15-4.22 (m, 8H,
Ɉɋɇ2), 6.24 (s, 2H, CH2), 7.76 (s, 1H, CH)
31
P NMR (121.49 MHz, CDCl3) δ: 22.4 (m, P(O)(OEt)2)
Calculated for C18H35N3O8P2: C, 44.75; H, 7.24; N, 8.70. Found: C, 44.67; H, 7.31; N,
8.49
- 128 -
Experimental section
Tetraethyl 2-[1-(2,3,4,6-tetra-O-acetyl-ȕ-D-glucopyranosyl)-1H-1,2,3-triazol-4-yl]
ethane-1,1-diyldipshopshonate (49)
Yield 66 %, colorless oil
1
H NMR (300.13 MHz, CDCl3) δ: 1.28-1.40 (m, 12H, CH3), 1.93 (s, 3H, ɋɇ3), 2.11 (d,
9H, CH3, 3JH-H =11.2 Hz), 2.89-3.09 (m, 1H, ɋɇ), 3.32-3.47 (m, 2H, CH2), 4.01-4.29
(m, 8H, Ɉɋɇ2, 3H), 5.28 (t, 1H, CH, 3JH-H =10.05 Hz), 5.45 (t, 1H, CH, 3JH-H = 9.12
Hz), 5.54 (t, 1H, CH, 3JH-H = 9.12 Hz), 5.89 (d, 1H, 3JH-H = 9.12 Hz), 7.76 (s, 1H, CH)
31
P NMR (121.49 MHz, CDCl3) δ: 22.33 (m, P(O)(OEt)2)
Calculated for C26H43N3O15P2: C, 44.64; H, 6.15; N, 6.01. Found: C, 44.15; H, 6.14;
N, 5.58.
Tetraethyl (2-{1-[3-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin1(2H)
-yl)tetrahydrofuran-2-yl]-1H-1,2,3-triazol-4-yl}ethane-1,1-
diyl)bis(phosphonate) (50)
Yield 92 %, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.31 (m, 12H, CH3), 1.96 (s, 3H, CH3), 2.97 (m, 3H,
CH2,CH), 3.37 (m, 3H, CH2,CH), 3.86 (dm, 1H, CH), 4.10 (dm, 1H, CH2), 4.17 (m,
8H, OCH2), 4.40 (m, 1H, CH2), 5.38 (m, 1H, CH), 6.23 (m, 1H, CH), 7.43 (s, 1H,
=CH), 7.75 (s, 1H, CH), 8.55 (br.s, 1H, NH)
31
P NMR (80.99 MHz, CDCl3) δ: 24.73 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 12.9 (CH3), 16.7 and 16.8 (CH3CH2O), 22.5 (CH2),
37.0 (t, CP, 1JCP= 133.6 Hz), 59.3 (CH-N), 60.8 (CH2OH), 63.3 (dd, OCH2, 2JCP =6.5
- 129 -
Experimental section
Hz), 85.5 (N-CH), 87.5 (CH-CH2OH), 111.2 (=C-CH3), 123.6 (N-CH=C), 137.6 (=CH2N), 145.5 and 145.6 and 145.7 (Car), 151.0 (C=O), 164.8 (C=O)
Calculated for C22H37N5O10P2: C, 44.48; H, 6.23; N, 11.79. Found: C, 44.35; H, 6.24;
N, 11.58
2-[1-(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)-1H-1,2,3-triazol-4-yl]ethane-1,1diyl] bis (phosphonic acid) (51)
1eq (0.27 mmol) of bisphosphonate was dissolved in dry chloroform and 7 eq (1.96
mmol) of trimethylsilyl bromide was added. The reaction mixture was stirred at room
temperature 12h. Then, solvent was evaporated under reduced pressure. The
residue was added dropwise to mixture of methanol and water (8.1) and stirred 5h at
room temperature. The solvent was evaporated under reduced pressure
Yield 91%, light violet crystals, 57-62oC
1
H NMR (200.13 MHz, CDCl3) δ: 2.41-2.93 (m, 3H, CHP+CH2), 3.40 (dt, 2H, CH2,
3
JHH =6.1 Hz , 3JHP = 16.1 Hz), 4.72 (t, 2H, CH2, 3JHH = 7.2 Hz ), 7.61 (s, 1H, CH), (m),
9.72 (s, OH)
31
P NMR (80.99 MHz, CDCl3) δ: 21.35 (m, P(O)(OEt)2)
19
F NMR (188.31 MHz, CDCl3) δ: -81.4 (m, 3F, CF3), -114.7 (m, 2F, CF2CH2), -
122.92 (m, 2F, CF2CF3), -123.88 (m, 2F, CH2CF2CF2CF2), -124.31 (m, 2F,
CH2CF2CF2), -126.9 (m, 2F, CF2CF2CF3)
IR (thin layer) ν/cm-1: 1027 (P-O-C), 1240 (P=O)
- 130 -
Experimental section
Tetraethyl [2-(1H-1,2,3-triazol-4-yl)ethane-1,1-diyl]bis(phosphonate) (52)
To a solution of tert-butyl{4-[2,2-bis(diethoxyphosphoryl)ethyl]-1H-1,2,3-triazol-1yl}acetate (0.48 g, 0.9 mmol) in MeOH (3.6 mL), NaOH (1M aq. solution, 3.6 mL) was
added. The reaction mixture was stirred at r.t. for 3h and subsequently neutralized
with 1N HCl (5 mL), diluted with H2O (20 mL) and extracted three times with ethyl
acetate (45 mL). The organic layer was dried over MgSO4 and evaporated under
vacuum to yield the product in pure form
Yield 65%, colorless oil
1
H NMR (300.13 MHz, CDCl3) δ: 1.35 (t, 12H, ɋɇ3, 3JH-H = 7.08 Hz), 2.93-3.01 (m,
1H, ɋɇ), 3.36-3.42 (m, 2H, CH2), 4.17-4.23 (m, 8H, Ɉɋɇ2), 5.77 (s, 1H, NH), 7.61 (d,
1H, CH, 3JHH =10.95Hz)
31
P NMR (121.49 MHz, CDCl3) δ: 22.4 (m, P(O)(OEt)2)
Calculated for C12H25N3O6P2: C, 39.05; H, 6.77; N, 11.39. Found: C, 38.69; H, 6.72;
N, 10.22
[2-(1H-1,2,3-Triazol-4-yl)ethane-1,1-diyl]bis(phosphonic acid) (53)
A solution of trimethylsilyl bromide (0.54 g) in 2 mL of CHCl3 was added dropwise to
a solution of Tetraethyl [2-(1H-1,2,3-triazol-4-yl)ethane-1,1-diyl]bis(phosphonate) (59)
(0.26 g) in 7 mL of CHCl3. A reaction mixture was allowed to stir at r.t. overnight, then
solvent was removed under reduced pressure (rotor evaporator) and the residue was
dissolved in 10 mL of methanol. After stirring for 1 h methanol was removed in
vacuum to give crude product, which was crystallized from the mixture
ethanol/hexane.
Yield 77%, white crystals m.p. 203-205 oC
- 131 -
Experimental section
1
H NMR (200.13 MHz, d6-DMSO) δ: 2.93-3.01 (m, 1H, ɋɇ), 3.36-3.42 (m, 2H, CH2),
5.77 (s, 1H, NH), 7.61 (br.s, 1H, CH)
31
P NMR (121.49 MHz, CDCl3) δ: 22.4 (m, P(O)(OEt)2)
Calculated for C4H9N3O6P2: C, 18.67; H, 3.50; N, 16.33. Found: C, 18.69; H, 3.52; N,
16.21
Typical procedure for the preparation of compounds 54, 55, 56
Method B
Organic azide (2.0 mmol) and Tetraethyl hepta-1,6-diyne-4,4-diyldiphosphonate (38)
(2.0 mmol) were suspended in 1:4 H2O-t-BuOH (8 mL). To this was added
CuSO4Â5H2O (5 M solution, 0.1 mmol, 5 mol%) and sodium ascorbate (0.6 mmol).
The mixture was stirred at r.t. for 24 h, at which time TLC (silica, petroleum etheracetone) indicated complete conversion. The resulted solution was concentrated
under reduced pressure (rotar evaporator). The residue was dissolved in 30 mL of
brine and then extracted with ethyl acetate (3 x 30 mL). Combined organic layers
were washed with 5 % aq NH4OH (2 x 10 mL), dried over MgSO4, filtered and solvent
was removed under vacuum to give analytically pure product.
Tetraethyl 1,3-bis(1benzyl-1H-1,2,3-triazol-4-yl)propane-2,2-diyldipshopshonate
(54)
Yield 88%, white crystals, m.p. 86-88 oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.33 (t, 12H, CH3, 3JH-H = 7.3 Hz), 3.32 (dd, 4H,
- 132 -
Experimental section
CH2, 3JHP= 16.0 Hz), 4.21-4.27 (m, 8H, OCH2), 5.52 (s, 4H, CH2), 7.30-7.35 (m,
10HAR), 7.91 (s, 2H, CH)
31
P NMR (80.99 MHz, CDCl3) δ: 25.1 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.6 (d, CH3, 3JCP = 3.3 Hz), 16.7 (d, CH3, 3JCP = 3.1
Hz), 26.6 (PCH2), 47.6 (t, CP, 1JCP= 130.9), 54.2 (NCH2), 63.1 & 63,2 (POCH2), 125.0
(CH=), 128.5 and 128.9 and 129.4 (CHAr), 135.6(ipso-CAr), 143.4 (C=)
HRMS calculated for C29H40N6O6P2 (M+) 630.2484, found 630.2480
Tetraethyl 1,3-bis[1-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-1H-1,2,3-triazol4yl]pro-pane-2,2-diyldiphosphonate (55)
Yield 92%, white crystals, m.p. 126-129 oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.30 (t, 12H, CH3, 3JH-H = 7.1Hz ), 2.91-2.95 (m, 4H,
CH2), 3.41 (dd, 4H, CH2, 3JHP = 16.0 Hz), 4.21-4.27 (m, 8H, OCH2), 4.73 (t, 4H, CH2,
3
JH-H = 7.4Hz ), 8.01 (s, 2H, CH)
19
F NMR (188.31 MHz, CDCl3) δ: -76.9 (3F, m, CF3), -110.3 (2F, m, CF2CH2), -117.9
(2F, m, CF2CF3), -118.9 (2F, m, CH2CF2CF2CF2), -119.6 (2F, m, CH2CF2CF2), -122.2
(2F, m, CF2CF2CF3)
31
P NMR (80.99 MHz, CDCl3) δ: 29.8 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.6 (d, CH3, 2JCP = 6.2 Hz), 26.5 , 30.4 (t, CH2CF2,
2
JCF = 21.9 Hz), 36.9 and 39.6 (t, CP, 1JCP= 133.1), 42.6 and 43.2, 63.4 (d, OCH2,
2
JCP = 7.2 Hz), 109.3 - 121.1, 125.6 (=C), 142.7 (C=)
Calculated for C31H34N6O6P2F26 C, 32.59; H, 3.00. Found: C, 32.28; H, 3.21.
- 133 -
Experimental section
Tetraethyl
1,3-bis(1glucopyranosyl-1H-1,2,3-triazol-4-yl)propane-2,2-
diyldipshopshonate (56)
Yield 85%, white crystals, m.p. 98-102 oC
1
H NMR (200.13 MHz, CDCl3) δ: 1.20 (t, 12H, CH3, 3JH-H = 6.9 Hz),1.90 (s, 6H, CH3),
2.00 (d, 18H, CH3, 3JH-H =8 Hz), 3.40 (dd, 4H, CH2, 3JHP = 16.0 Hz), 3.58-3.75 (m, 2H,
ɋɇ), 4.14-4.20 (m, 8H, OCH2), 5.21 (t, 2H, CH, 3JH-H =9.61 Hz), 5.37 (t, 2H, CH, 3JH-H
= 9.25 Hz), 5.55 (t, 2H, CH, 3JH-H = 9.32 Hz), 5.85 (d, 2H, 3JH-H = 9.38 Hz), 8.01 (s,
2H, CH)
31
P NMR (80.99 MHz, CDCl3) δ: 24.9 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.6 (d, CH3, 3JCP = 3.2 Hz), 16.7 (d, CH3, 3JCP = 3.3
Hz), 20.7 (s, CH2COCH3), 21.1 (s, COCH3), 26.8 (PCH2), 47.2 (t, CP, 1JCP= 131.0),
60.7 (d, OCH2, 2JCP = 7.0 Hz), 68.1(s, CH2COCH3), 70.3 (s, CH), 73.5 (s, CH), 75.4
(CH-CH2), 85.9 (s, CH-N), 123.5 (=C), 140.7 (C=), 169.3 (CO), 169.8 (CO), 170.4
(CO), 170.9 (CO)
MS: (EI, 70eV, 200oC) m/z (%) 1082 [M]+, 1023 [M - 59]+, 973 [M - 109]+, 808 [M 274]+, and other fragments.
Typical procedure for the synthesis of triazols in situ 57- 60
A mixture of aromatic azide (1 mmol) and sodium azide (1 mmol) was suspended in
1:4 H2O-t-BuOH (8 mL) in stirred 12 h at room temperature. The conversion of halide
into azide was controlled by TLC (silica, petroleum ether-acetone). Then, to this
mixture was added tetraethyl but-3-yne-1,1-diyldiphosphonate (37) (1 mmol),
CuSO4Â5H2O (5 M solution, 0.1 mmol, 5 mol%) and sodium ascorbate (0.6 mmol).
The mixture was stirred r.t. for the next 12 h, at which time TLC (silica, petroleum
ether-acetone) indicated complete conversion. The reaction mixture was was filtered
- 134 -
Experimental section
off from participating inorganic salts. The residue was dissolved in 30 mL of brine and
then extracted with ethyl acetate (3 x 30 mL). Combined organic layers were washed
with 5 % aq NH4OH (2 x 10 mL), dried over MgSO4, filtered and solvent was removed
under vacuum to give analytically pure product.
Tetraethyl 2-(1-benzyl-1H-1,2,3-triazol-4-yl)ethane-1,1-diyldipshopshonate (57)
Yield 77%, colorless oil
1
H NMR (200.13 MHz, CDCl3) δ: 1.31 (t, 12H, CH3, 3JHH = 6.8Hz), 2.90 (tt, 1H, CHP,
3
JHH = 6.7, 2JHP = 24.0 Hz), 3.33 (dt, 2H, CH2, 3JHH = 6.4, 3JHP = 15.9 Hz), 4.21-4.26
(m, 8H, OCH2), 5.52 (s, 2H, CH2), 7.31-7.33 (m, 5HAR), 7.55 (s, 1H, CH)
31
P NMR (80.99 MHz, CDCl3) δ: 23.6 (m, P(O)(OEt)2)
13
C NMR (50.32 MHz, CDCl3) δ: 16.7 (CH3), 23.6, 37.2 (t, CP, 1JCP = 133.0 Hz), 55.2
(NCH2), 62.9 (d, OCH2, 2JCP =6.5 Hz), 63.1 (d, OCH2, 2JCP =6.2 Hz), 122.6 (ɋɇ=),
128.5 and 128.6 and 129.4 (CAr), 135.3 (CAr), 145.9 (C=)
Octaethyl
2,2’-[1,1’-(1,4-phenylenebis(methylene)]
[bis(1H-1,2,3-triazol-4,1-
diyl)]bis(ethane-2,1,1-triyl)tetraphosphonate (58)
Yield 67%, colorless oil
1
H NMR (200.13 MHz, CD3CN) δ: 1.13-1.18 (m, CH3, 24H, 3JH-H = 7.1 Hz), 3.00-3.05
(m, CHP+ CH2, 6H), 3.85- 4.04 (m, OCH2, 16H), 5.48 (s, CH2, 4H), 7.32 (s, HAR, 4H),
7.63 (s, Hc, 2H)
31
P NMR (80.99 MHz, CD3CN) δ: 29.3 (m, P(O)(OEt)2)
- 135 -
Experimental section
13
C NMR (50.32 MHz, CDCl3) δ: 16.1 (d, CH3, 2JCP = 6.1 Hz), 22.0 (PCH2), 37.1 (t,
CP, 1JCP = 133.8 Hz), 54.2 (NCH2), 63.2 (d, OCH2, 2JCP =6.4 Hz), 63.4 (d, OCH2, 2JCP
=6.3 Hz), 120.7 (ɋɇ=), 129.3 (CAr), 136.3 (CAr), 149.6 (C=)
Calculated for C32H56Br2N6O12P4Na2: C, 36.68; H, 5.35. Found: C, 37.11; H, 5.55
Octaethyl
2,2’-[1,1’-(1,3-phenylenebis(methylene))bis(1H-1,2,3-triazol-4,1-
diyl))bis(eth-ane-2,1,1-triyl]tetraphosphonate (59)
Yield 65%, colorless oil
1
H NMR (200.13 MHz, CD3CN) δ: 1.31 (m, CH3, 24H, 3JH-H = 7.3 Hz), 2.95 (m, CHP+
CH2, 6H), 3.82-4.13 (m, OCH2, 16H), 5.50 (s, CH2, 4H), 7.32 (s, HAR, 4H), 7.65 (s, Hc,
2H)
31
P NMR (80.99 MHz, CD3CN) δ: 24.0 (m, P(O)(OEt)2)
Calculated for C32H56Br2N6O12P4Na2: C, 36.68; H, 5.35. Found: C, 37.18; H, 5.73
Octaethyl
2,2’-[1,1’-(1,3-phenylenebis(methylene)-2,4,5,6-tetrafluoro))bis(1H-
1,2,3-triazol-4,1-diyl))bis(eth-ane-2,1,1- triyl]tetraphosphonate (60)
Yield 68%, colorless oil
1
H NMR (200.13 MHz, CD3Cl) δ: 1.26 (m, CH3, 24H, 3JH-H = 7.0 Hz), 2.95 (tt, CHP,
2H, 3JHH = 6.5, 2JHP = 23.4 Hz), 3.30 (dt, 4H, CH2, 3JHH = 6.4, 3JHP = 15.0 Hz), 4.10
(m, OCH2, 16H), 4.48 (s, CH2, 4H), 7.59 (s, Hc, 2H)
19
F NMR (188.31 MHz, CDCl3) δ: -142.72
31
P NMR (80.99 MHz, CD3Cl) δ: 23.37 (m, P(O)(OEt)2)
- 136 -
Experimental section
IR (thin layer) ν/cm-1: 1026 (P-O-C), 1241 (P=O)
General procedure for the preparation of fluorinated bisphosphonates
2-chloro, 2,2-difluoroacetyl chloride (6.7mmol) was dissolved in dry monoglyme (100
mL) and stirred. Flask was cooled down to 0oC. Next, tris (trimethylsilyl) phosphite or
triethyl phosphite (13.4 mmol) were added dropwise. The reaction mixture was stirred
at room temperature over a period of 0.5 h. Then, the mixture was heated at 40oC for
4 hours. The solvent was removed under reduced pressure and the crude product
was purified by distillation to give analitically pure.
Tetrakis (trimethylsilyl) 3,3-difluoroprop-2-en-1,2 diylbis(phosphonate) (64)
Yield 84%, colorless oil. B.p: 62oC/0.005 mmHg
1
H NMR (200.13 MHz, CDCl3) δ: 0.2 (s, CH3, 36H)
31
3
JPFcis= 6.5 Hz), -20.7 (ddd, 1P, 4JPFtrans = 6.5 Hz, 4JPFcis = 8.7 Hz)
19
4
P NMR (80.99 MHz, CDCl3) δ: -12.71 (ddd, 1P, 2JPP = 24.3 Hz, 3JPFtrans = 17.7 Hz,
F NMR (188.31 MHz, CDCl3) δ: -79.6 (ddd, 1F, 2JFF = 25.0 Hz, 3JPFcis = 6.5 Hz,
JPFtrans= 6.5 Hz), -94.1 (ddd, 1F, 2JFF = 26.0 Hz, 3JPFtrans = 17.5 Hz, 4JPFtrans = 8.9 Hz).
13
C NMR (50.32 MHz, CDCl3) δ: 1.3 (d, CH3, 3JCP = 26.5 Hz), 109.4 (dddd, PCOP,
1
JCP = 250.4 Hz, 2JCP = 39.8 Hz), 115.5 (dddd, CF2, JCF = 295.3 Hz, 2JCP = 39.8 Hz,
3
JCP = 26.4 Hz)
Calculated for C14H36F2O7P2Si4: C, 31.80; H, 6.86; P, 11.72. Found: C, 31.44; H,
7.00; N, 11.79
- 137 -
Experimental section
Tetraethyl 3, 3-difluoroprop-2-en-1,2-diylbis(phosphonate) (65)
Yield 73%, colorless oil. Sdp: 48oC/0.005 mmHg
1
H NMR (200.13 MHz, CDCl3) δ: 1.17 (t, CH3, 12H, 2JHH = 7.02 Hz), 3.99 (q, OCH2,
8H)
31
P NMR (80.99 MHz, CDCl3) δ: 6.48 (m), -3.15 (dm, 3JPCOP = 6.23 Hz)
19
F NMR (188.31 MHz, CDCl3) δ: -77.2 (ddd, 1F, 2JFF = 26.6 Hz, 3JPFcis = 18.0 Hz,
4
JPFtrans= 8.6 Hz), -92.1 (ddd, 1F, 2JFF = 25.0 Hz, 3JPFcis = 15.0 Hz, 4JPFtrans = 4.5 Hz)
Calculated for C10H20F2O7P2: C, 34.10; H, 5.72; P, 17.59. Found: C, 33.96; H, 5.82;
P, 17.32. M (+) 352
3, 3-difluoroprop-2-en-1,2-diyldiphosphonic acid (66)
Tetrakis (trimethylsilyl) 3,3-difluoroprop-2-en-1,2 diylbis(phosphonate) (1.98 mmol)
was put into the flask . Then, 2 mL of methanol and 1 ml of water were added. The
reaction mixture was stirred at room temperature overnight. After this period of time,
some drops of ethyl acetate were added. The reaction mixture was extracted with
ether (3 x 20 mL). The combined organic layers were washed with brine (25 mL),
dried over Na2SO4 and filtered. The solvent was removed under reduced pressure to
give analytically pure fluorinated bisphosphonate.
Yield 84%, white crystals, m.p. 101-105 oC
1
H NMR (200.13 MHz, CDCl3) δ: 5.46 (s, OH)
- 138 -
Experimental section
31
P NMR (80.99 MHz, CDCl3) δ: -3.61 (m)
19
F NMR (188.31 MHz, CDCl3) δ: -91.6 (m, 1F, 2JFF = 20.0 Hz, 3JPFcis = 13.4 Hz,
4
JPFtrans= 7.8 Hz), -104.7 (m, 1F, 2JFF = 20.0 Hz, 3JPFtrans = 14.8 Hz, 4JPFcis = 10.1 Hz)
MS: (ESI negativ, CH3CN) 238.7 [M-H]-, 478.4 [2M-H]-, 718.3 [2M-H]-, (ESI positiv,
CH3CN) 240.8 [M+H]+, 480.6 [2M+H]+
- 139 -
X-ray data
Chapter 5
X-ray data
Methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(1H-indol-3-yl)alaninate (17)
Table 1
Crystal data and structure refinement for 17
Identification code
hs3
Empirical formula
C16 H15 F3 N2 O5
Formula weight
372.30
Temperature
173(2) K
Wavelength
71.073 pm
- 140 -
X-ray data
Crystal system
Triclinic
Space group
P -1
Unit cell dimensions
a = 799.5(3) pm
Į = 112.74(3)°
b = 955.7(5) pm
ȕ = 95.66(3)°
c = 1204.3(4) pm
Ȗ = 103.69(2)°
3
Volume
0.8058(6) nm
Z
2
Density (calculated)
1.534 Mg/m
Absorption coefficient
0.136 mm-1
F(000)
384
Crystal size
0.7 x 0.5 x 0.2 mm
Theta range for data collection
2.68 to 27.50°.
Reflections collected
4278
Independent reflections
3465 [R(int) = 0.0336]
Completeness to theta = 27.50°
93.4 %
Absorption correction
None
Refinement method
Full-matrix least-squares on F
Data / restraints / parameters
3465 / 0 / 248
Goodness-of-fit on F
2
3
3
1.149
Final R indices [I>2sigma(I)]
R1 = 0.0956, wR2 = 0.3306
R indices (all data)
R1 = 0.1156, wR2 = 0.3458
Largest diff. peak and hole
-3
0.738 and -0.493 e.Å
- 141 -
2
X-ray data
Table 2
4
2
Atomic coordinates (x 10 ) and equivalent isotropic displacement parameters (pm x
-1
10 ) for hs3. U(eq) is defined as one third of the trace of the orthogonalized Uij
tensor.
x
y
z
U(eq)
C(1)
3275(6)
1870(6)
6413(5)
25(1)
C(2)
4126(6)
1451(6)
7293(4)
21(1)
C(3)
4103(7)
-162(6)
6641(5)
27(1)
N(2)
3250(6)
-662(5)
5441(4)
32(1)
C(4)
2761(8)
544(6)
5307(5)
32(1)
C(5)
3039(6)
3483(6)
6726(5)
24(1)
C(6)
2179(7)
3674(7)
5613(5)
29(1)
F(1)
644(5)
2586(5)
5027(3)
42(1)
F(2)
1941(5)
5103(4)
5963(3)
43(1)
F(3)
3229(5)
3563(5)
4791(3)
40(1)
C(7)
1869(7)
3819(6)
7695(5)
27(1)
O(2)
2326(6)
4990(5)
8654(4)
40(1)
O(3)
370(5)
2672(5)
7341(4)
32(1)
C(8)
-780(8)
2857(8)
8210(6)
41(2)
N(1)
4708(6)
4773(5)
7309(4)
26(1)
C(9)
6284(7)
4763(6)
7027(4)
24(1)
O(4)
6624(5)
3760(4)
6170(4)
34(1)
C(10)
7718(6)
6285(6)
7968(5)
24(1)
O(5)
7459(6)
7115(5)
8922(4)
42(1)
O(6)
9178(5)
6468(5)
7559(4)
32(1)
C(11)
10636(7)
7882(7)
8356(6)
35(1)
C(12)
10427(8)
9328(7)
8218(6)
40(1)
C(13)
4863(7)
2222(6)
8562(5)
29(1)
C(14)
5546(7)
1412(7)
9115(5)
34(1)
C(15)
5554(7)
-181(7)
8445(6)
35(1)
C(16)
4814(8)
-967(7)
7202(6)
36(1)
- 142 -
X-ray data
Table 3
Bond lengths [pm] and angles [°] for hs3
C(1)-C(4)
137.5(7)
C(7)-O(2)
120.1(7)
C(1)-C(2)
143.6(7)
C(7)-O(3)
131.8(6)
C(1)-C(5)
150.2(7)
O(3)-C(8)
144.7(6)
C(2)-C(13)
140.4(7)
N(1)-C(9)
133.8(7)
C(2)-C(3)
142.8(7)
C(9)-O(4)
121.3(6)
C(3)-N(2)
137.8(8)
C(9)-C(10)
154.7(7)
C(3)-C(16)
138.1(9)
C(10)-O(5)
118.8(6)
N(2)-C(4)
136.1(8)
C(10)-O(6)
131.1(6)
C(5)-N(1)
147.0(6)
O(6)-C(11)
146.1(6)
C(5)-C(6)
153.9(8)
C(11)-C(12)
149.8(9)
C(5)-C(7)
155.2(7)
C(13)-C(14)
136.7(9)
C(6)-F(1)
132.0(6)
C(14)-C(15)
142.3(9)
C(6)-F(2)
133.1(7)
C(15)-C(16)
137.8(9)
C(6)-F(3)
135.0(6)
C(4)-C(1)-C(2)
106.9(5)
F(1)-C(6)-C(5)
112.7(4)
C(4)-C(1)-C(5)
129.6(5)
F(2)-C(6)-C(5)
110.8(4)
C(2)-C(1)-C(5)
123.5(4)
F(3)-C(6)-C(5)
110.7(4)
C(13)-C(2)-C(3)
118.2(5)
O(2)-C(7)-O(3)
125.5(5)
C(13)-C(2)-C(1)
135.3(5)
O(2)-C(7)-C(5)
122.5(5)
C(3)-C(2)-C(1)
106.5(4)
O(3)-C(7)-C(5)
112.0(4)
N(2)-C(3)-C(16)
130.4(5)
C(7)-O(3)-C(8)
115.9(4)
N(2)-C(3)-C(2)
106.9(5)
C(9)-N(1)-C(5)
127.2(4)
C(16)-C(3)-C(2)
122.7(5)
O(4)-C(9)-N(1)
127.8(5)
C(4)-N(2)-C(3)
109.9(4)
O(4)-C(9)-C(10)
122.2(5)
N(2)-C(4)-C(1)
109.8(5)
N(1)-C(9)-C(10)
110.0(4)
N(1)-C(5)-C(1)
112.6(4)
O(5)-C(10)-O(6)
127.5(5)
N(1)-C(5)-C(6)
107.4(4)
O(5)-C(10)-C(9)
122.0(5)
C(1)-C(5)-C(6)
114.0(4)
O(6)-C(10)-C(9)
110.5(4)
N(1)-C(5)-C(7)
105.1(4)
C(10)-O(6)-C(11)
116.1(4)
C(1)-C(5)-C(7)
109.6(4)
O(6)-C(11)-C(12)
111.3(5)
C(6)-C(5)-C(7)
107.8(4)
C(14)-C(13)-C(2)
118.9(5)
F(1)-C(6)-F(2)
108.4(5)
C(13)-C(14)-C(15)
122.2(5)
F(1)-C(6)-F(3)
107.4(4)
C(16)-C(15)-C(14)
119.8(6)
F(2)-C(6)-F(3)
106.6(4)
C(15)-C(16)-C(3)
118.3(5)
- 143 -
X-ray data
Table 4
Anisotropic
displacement
parameters
displacement factor exponent takes the form: -2
U11
U22
U33
C(1)
22(2)
20(2)
24(2)
C(2)
18(2)
18(2)
C(3)
25(2)
N(2)
-1
10 )
2
(pm x
2
2
for
hs3.
2 11
+ ... + 2 h k a* b* U
[ h a* U
U23
The
anisotropic
12
]
U13
U12
5(2)
7(2)
-1(2)
22(2)
3(2)
9(2)
2(2)
20(2)
31(3)
5(2)
15(2)
3(2)
35(2)
17(2)
30(2)
-1(2)
12(2)
0(2)
C(4)
34(3)
23(2)
27(2)
3(2)
8(2)
1(2)
C(5)
18(2)
22(2)
27(2)
7(2)
8(2)
1(2)
C(6)
28(3)
28(3)
29(3)
10(2)
7(2)
5(2)
F(1)
30(2)
50(2)
33(2)
15(2)
-2(1)
-5(2)
F(2)
50(2)
41(2)
43(2)
20(2)
13(2)
19(2)
F(3)
41(2)
52(2)
32(2)
22(2)
17(2)
13(2)
C(7)
27(3)
24(2)
29(2)
9(2)
11(2)
7(2)
O(2)
38(2)
34(2)
32(2)
0(2)
17(2)
4(2)
O(3)
25(2)
33(2)
30(2)
9(2)
15(2)
2(2)
C(8)
38(3)
47(4)
45(3)
22(3)
29(3)
13(3)
N(1)
25(2)
20(2)
24(2)
3(2)
9(2)
2(2)
C(9)
27(2)
17(2)
22(2)
6(2)
5(2)
4(2)
O(4)
35(2)
22(2)
34(2)
2(2)
18(2)
1(2)
C(10)
21(2)
20(2)
26(2)
6(2)
5(2)
4(2)
O(5)
36(2)
32(2)
34(2)
-3(2)
12(2)
-5(2)
O(6)
22(2)
26(2)
39(2)
5(2)
11(2)
2(2)
C(11)
21(2)
33(3)
41(3)
9(2)
3(2)
1(2)
C(12)
33(3)
29(3)
46(3)
10(3)
5(3)
0(2)
C(13)
30(3)
28(3)
24(2)
6(2)
12(2)
5(2)
C(14)
30(3)
41(3)
30(3)
14(2)
14(2)
9(2)
C(15)
27(3)
40(3)
49(3)
26(3)
17(2)
10(2)
C(16)
31(3)
27(3)
50(3)
13(2)
25(3)
11(2)
- 144 -
X-ray data
Table 5
4
2
Hydrogen coordinates (x 10 ) and isotropic displacement parameters (pm x 10
hs3
x
H(2)
3100(80)
H(4)
2156
H(8A)
y
-1550(80)
z
U(eq)
4770(60)
26(15)
479
4561
37(8)
-244
2753
8931
61(10)
H(8B)
-1919
2033
7818
61(10)
H(8C)
-955
3909
8469
61(10)
H(1A)
4770(80)
5450(80)
8120(60)
30(16)
H(11A)
11759
7714
8143
43(14)
H(11B)
10684
8052
9224
43(14)
H(12A)
10342
9149
7354
61(10)
H(12B)
11450
10245
8731
61(10)
H(12C)
9355
9536
8480
61(10)
H(13)
4885
3289
9029
37(8)
H(14)
6033
1930
9975
37(8)
H(15)
6068
-701
8853
37(8)
H(16)
4794
-2036
6744
37(8)
- 145 -
-1
) for
X-ray data
Table 6
Torsion angles [°] for hs3
C(4)-C(1)-C(2)-C(13)
-176.8(5)
C(5)-C(7)-O(3)-C(8)
177.8(5)
C(5)-C(1)-C(2)-C(13)
2.8(8)
C(1)-C(5)-N(1)-C(9)
38.6(7)
C(4)-C(1)-C(2)-C(3)
0.9(5)
C(6)-C(5)-N(1)-C(9)
-87.7(6)
C(5)-C(1)-C(2)-C(3)
-179.5(4)
C(7)-C(5)-N(1)-C(9)
157.8(5)
C(13)-C(2)-C(3)-N(2)
177.3(4)
C(5)-N(1)-C(9)-O(4)
7.5(9)
C(1)-C(2)-C(3)-N(2)
-0.9(5)
C(5)-N(1)-C(9)-C(10)
-173.3(5)
C(13)-C(2)-C(3)-C(16)
-1.8(7)
O(4)-C(9)-C(10)-O(5)
-165.8(6)
C(1)-C(2)-C(3)-C(16)
-180.0(5)
N(1)-C(9)-C(10)-O(5)
14.9(8)
C(16)-C(3)-N(2)-C(4)
179.6(5)
O(4)-C(9)-C(10)-O(6)
13.3(7)
C(2)-C(3)-N(2)-C(4)
0.6(6)
N(1)-C(9)-C(10)-O(6)
-165.9(4)
C(3)-N(2)-C(4)-C(1)
-0.1(6)
O(5)-C(10)-O(6)-C(11)
-2.0(9)
C(2)-C(1)-C(4)-N(2)
-0.5(6)
C(9)-C(10)-O(6)-C(11)
179.0(4)
C(5)-C(1)-C(4)-N(2)
179.8(5)
C(10)-O(6)-C(11)-C(12)
-80.4(6)
C(4)-C(1)-C(5)-N(1)
-126.2(6)
C(3)-C(2)-C(13)-C(14)
1.0(7)
C(2)-C(1)-C(5)-N(1)
54.2(6)
C(1)-C(2)-C(13)-C(14)
178.5(5)
C(4)-C(1)-C(5)-C(6)
-3.6(7)
C(2)-C(13)-C(14)-C(15)
0.7(8)
C(2)-C(1)-C(5)-C(6)
176.8(4)
C(13)-C(14)-C(15)-C(16)
-1.7(8)
C(4)-C(1)-C(5)-C(7)
117.2(6)
C(14)-C(15)-C(16)-C(3)
0.8(8)
C(2)-C(1)-C(5)-C(7)
-62.4(6)
N(2)-C(3)-C(16)-C(15)
N(1)-C(5)-C(6)-F(1)
-178.9(4)
C(2)-C(3)-C(16)-C(15)
C(1)-C(5)-C(6)-F(1)
55.7(6)
C(7)-C(5)-C(6)-F(1)
-66.2(6)
N(1)-C(5)-C(6)-F(2)
-57.2(5)
C(1)-C(5)-C(6)-F(2)
177.3(4)
C(7)-C(5)-C(6)-F(2)
55.5(5)
N(1)-C(5)-C(6)-F(3)
60.8(5)
C(1)-C(5)-C(6)-F(3)
-64.7(5)
C(7)-C(5)-C(6)-F(3)
173.5(4)
N(1)-C(5)-C(7)-O(2)
4.6(7)
C(1)-C(5)-C(7)-O(2)
125.8(6)
C(6)-C(5)-C(7)-O(2)
-109.7(6)
N(1)-C(5)-C(7)-O(3)
-174.4(4)
C(1)-C(5)-C(7)-O(3)
-53.2(6)
C(6)-C(5)-C(7)-O(3)
71.3(6)
O(2)-C(7)-O(3)-C(8)
-1.2(9)
- 146 -
-178.0(5)
0.9(8)
X-ray data
Methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2-(2-furyl)alaninate (20)
Table 1
Crystal data and structure refinement for 20
Identification code
hs1
Empirical formula
C12 H12 F3 N O6
Formula weight
323.23
Temperature
173(2) K
Wavelength
71.073 pm
Crystal system
Triclinic
Space group
P -1
Unit cell dimensions
a = 848.0(4) pm
Į = 72.84(2)°
b = 931.3(4) pm
ȕ = 66.46(2)°
c = 1021.7(5) pm
Ȗ = 76.69(2)°
Volume
0.7010(6) nm
Z
2
- 147 -
3
X-ray data
3
Density (calculated)
1.531 Mg/m
Absorption coefficient
0.147 mm-1
F(000)
332
Crystal size
1.0 x 0.8 x 0.7 mm
Theta range for data collection
2.64 to 27.50°.
Reflections collected
6432
Independent reflections
3194 [R(int) = 0.0274]
Completeness to theta = 27.50°
99.4 %
Absorption correction
None
Refinement method
Full-matrix least-squares on F
Data / restraints / parameters
3194 / 0 / 206
Goodness-of-fit on F
2
3
1.051
Final R indices [I>2sigma(I)]
R1 = 0.0426, wR2 = 0.1169
R indices (all data)
R1 = 0.0461, wR2 = 0.1203
Extinction coefficient
0.081(7)
Largest diff. peak and hole
-3
0.345 and -0.303 e.Å
Table 2
2
4
2
Atomic coordinates (x 10 ) and equivalent isotropic displacement parameters (pm x
-1
10 ) for hs1. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor
x
y
z
U(eq)
F(1)
4657(1)
3295(1)
1036(1)
44(1)
F(2)
6348(1)
1415(1)
1799(1)
45(1)
F(3)
7303(1)
3545(1)
547(1)
47(1)
O(1)
8203(1)
3911(1)
2888(1)
34(1)
O(2)
3227(1)
1784(1)
4076(1)
38(1)
O(3)
3269(1)
3350(1)
5365(1)
31(1)
- 148 -
X-ray data
Table 3
O(4)
2299(1)
5462(1)
2836(1)
34(1)
O(5)
5125(1)
8101(1)
1927(1)
35(1)
O(6)
2401(1)
8508(1)
1919(1)
30(1)
N(1)
5125(2)
5127(1)
2648(1)
27(1)
C(1)
6923(2)
2989(1)
3546(2)
25(1)
C(2)
7317(2)
1802(2)
4539(2)
30(1)
C(3)
8960(2)
1985(2)
4517(2)
32(1)
C(4)
9431(2)
3265(2)
3522(2)
35(1)
C(5)
5426(2)
3483(1)
2999(1)
25(1)
C(6)
5928(2)
2922(2)
1581(2)
34(1)
C(7)
3815(2)
2771(1)
4198(2)
27(1)
C(8)
1802(2)
2703(2)
6578(2)
39(1)
C(9)
3612(2)
5952(1)
2579(1)
25(1)
C(10)
3813(2)
7663(1)
2106(1)
26(1)
C(11)
2476(2)
10153(2)
1491(2)
36(1)
C(12)
994(2)
10897(2)
983(2)
44(1)
Bond lengths [pm] and angles [°] for hs1
F(1)-C(6)
133.60(18)
C(1)-C(2)
134.2(2)
F(2)-C(6)
133.96(18)
C(1)-C(5)
150.99(19)
F(3)-C(6)
133.37(19)
C(2)-C(3)
143.3(2)
O(1)-C(1)
136.88(17)
C(2)-H(2)
95.00
O(1)-C(4)
137.32(18)
C(3)-C(4)
133.9(2)
O(2)-C(7)
119.89(17)
C(3)-H(3)
95.00
O(3)-C(7)
132.28(18)
C(4)-H(4)
95.00
O(3)-C(8)
145.60(18)
C(5)-C(6)
155.1(2)
O(4)-C(9)
120.20(17)
C(5)-C(7)
155.19(19)
O(5)-C(10)
120.02(17)
C(8)-H(8A)
98.00
O(6)-C(10)
132.21(17)
C(8)-H(8B)
98.00
O(6)-C(11)
147.30(17)
C(8)-H(8C)
98.00
N(1)-C(9)
135.20(17)
C(9)-C(10)
155.17(19)
N(1)-C(5)
145.14(17)
C(11)-C(12)
149.6(2)
N(1)-H(1)
82.9(18)
C(11)-H(11A)
99.00
- 149 -
X-ray data
C(11)-H(11B)
99.00
C(12)-H(12B)
98.00
C(12)-H(12A)
98.00
C(12)-H(12C)
98.00
C(1)-O(1)-C(4)
106.05(11)
C(3)-C(4)-O(1)
110.35(13)
C(7)-O(3)-C(8)
114.59(11)
C(3)-C(4)-H(4)
124.8
C(10)-O(6)-C(11)
114.85(11)
O(1)-C(4)-H(4)
124.8
C(9)-N(1)-C(5)
124.45(12)
O(6)-C(11)-C(12)
C(9)-N(1)-H(1)
118.1(12)
O(6)-C(11)-H(11A)
110.2
C(5)-N(1)-H(1)
117.4(12)
C(12)-C(11)-H(11A)
110.2
C(2)-C(1)-O(1)
110.76(12)
O(6)-C(11)-H(11B)
110.2
C(2)-C(1)-C(5)
134.07(12)
C(12)-C(11)-H(11B)
110.2
O(1)-C(1)-C(5)
115.15(11)
H(11A)-C(11)-H(11B) 108.5
C(1)-C(2)-C(3)
106.07(13)
C(11)-C(12)-H(12A)
109.5
109.5
107.41(13)
C(1)-C(2)-H(2)
127.0
C(11)-C(12)-H(12B)
C(3)-C(2)-H(2)
127.0
H(12A)-C(12)-H(12B) 109.5
C(4)-C(3)-C(2)
C(11)-C(12)-H(12C)
106.77(13)
109.5
C(4)-C(3)-H(3)
126.6
H(12A)-C(12)-H(12C) 109.5
C(2)-C(3)-H(3)
126.6
H(12B)-C(12)-H(12C) 109.5
2
-1
Anisotropic displacement parameters (pm x 10 ) for hs1.
Table 4
displacement factor exponent takes the form: -2
2
2
2
[h a* U
The anisotropic
11 + ... + 2 h k a* b*
12
U ]
U11
U22
U33
U23
U13
U12
F(1)
50(1)
47(1)
48(1)
-18(1)
-30(1)
4(1)
F(2)
53(1)
32(1)
54(1)
-23(1)
-24(1)
10(1)
F(3)
45(1)
57(1)
34(1)
-14(1)
-6(1)
-6(1)
O(1)
27(1)
27(1)
46(1)
-2(1)
-17(1)
-5(1)
O(2)
38(1)
28(1)
53(1)
-11(1)
-15(1)
-10(1)
O(3)
27(1)
30(1)
34(1)
-7(1)
-7(1)
-7(1)
O(4)
27(1)
26(1)
49(1)
-7(1)
-16(1)
-3(1)
O(5)
30(1)
25(1)
48(1)
-1(1)
-15(1)
-5(1)
- 150 -
X-ray data
Table 5
O(6)
25(1)
22(1)
37(1)
-5(1)
-10(1)
2(1)
N(1)
24(1)
19(1)
37(1)
-3(1)
-12(1)
-3(1)
C(1)
22(1)
22(1)
32(1)
-9(1)
-8(1)
-2(1)
C(2)
30(1)
24(1)
34(1)
-7(1)
-12(1)
0(1)
C(3)
30(1)
30(1)
37(1)
-12(1)
-16(1)
6(1)
C(4)
26(1)
34(1)
49(1)
-13(1)
-18(1)
-1(1)
C(5)
25(1)
20(1)
30(1)
-6(1)
-11(1)
-1(1)
C(6)
37(1)
31(1)
37(1)
-12(1)
-17(1)
2(1)
C(7)
24(1)
20(1)
36(1)
-5(1)
-14(1)
-1(1)
C(8)
25(1)
21(1)
28(1)
-5(1)
-9(1)
-1(1)
C(10)
25(1)
21(1)
27(1)
-4(1)
-8(1)
0(1)
C(11)
32(1)
21(1)
44(1)
-2(1)
-10(1)
1(1)
C(12)
50(1)
33(1)
47(1)
-10(1)
-24(1)
13(1)
4
2
Hydrogen coordinates (x 10 ) and isotropic displacement parameters (pm x 10
hs1
x
y
z
U(eq)
H(1)
5930(20)
5590(20)
2500(19)
26(4)
H(2)
6642
1003
5136
35
H(3)
9593
1326
5096
38
H(4)
10468
3668
3288
42
H(8A)
789
2941
6280
58
H(8B)
1539
3133
7421
58
H(8C)
2091
1602
6848
58
H(11A)
3590
10387
693
43
H(11B)
2379
10524
2338
43
H(12A)
1099
10516
151
66
H(12B)
1012
11996
681
66
H(12C)
-102
10667
1786
66
- 151 -
-1
) for
X-ray data
Table 6
Torsion angles [°] for hs1
C(4)-O(1)-C(1)-C(2)
-0.67(16)
C(11)-O(6)-C(10)-O(5)
-2.0(2)
C(4)-O(1)-C(1)-C(5)
-179.48(11)
C(11)-O(6)-C(10)-C(9)
179.06(11)
O(1)-C(1)-C(2)-C(3)
0.26(16)
O(4)-C(9)-C(10)-O(5)
176.46(13)
C(5)-C(1)-C(2)-C(3)
178.76(13)
N(1)-C(9)-C(10)-O(5)
-3.67(18)
C(1)-C(2)-C(3)-C(4)
0.26(16)
O(4)-C(9)-C(10)-O(6)
-4.51(19)
C(2)-C(3)-C(4)-O(1)
-0.68(17)
N(1)-C(9)-C(10)-O(6)
175.36(11)
C(1)-O(1)-C(4)-C(3)
0.84(16)
C(10)-O(6)-C(11)-C(12)
166.60(12)
C(9)-N(1)-C(5)-C(1)
-158.07(12)
C(9)-N(1)-C(5)-C(6)
83.82(16)
C(9)-N(1)-C(5)-C(7)
-37.88(18)
C(2)-C(1)-C(5)-N(1)
146.42(15)
O(1)-C(1)-C(5)-N(1)
-35.13(15)
C(2)-C(1)-C(5)-C(6)
-95.58(18)
O(1)-C(1)-C(5)-C(6)
82.87(14)
C(2)-C(1)-C(5)-C(7)
24.1(2)
O(1)-C(1)-C(5)-C(7)
-157.50(11)
N(1)-C(5)-C(6)-F(3)
56.25(15)
C(1)-C(5)-C(6)-F(3)
-61.61(14)
C(7)-C(5)-C(6)-F(3)
179.44(10)
N(1)-C(5)-C(6)-F(1)
-63.08(15)
C(1)-C(5)-C(6)-F(1)
179.06(11)
C(7)-C(5)-C(6)-F(1)
60.10(15)
N(1)-C(5)-C(6)-F(2)
175.53(12)
C(1)-C(5)-C(6)-F(2)
57.67(15)
C(7)-C(5)-C(6)-F(2)
-61.28(15)
C(8)-O(3)-C(7)-O(2)
-0.74(19)
C(8)-O(3)-C(7)-C(5)
-177.51(11)
N(1)-C(5)-C(7)-O(2)
130.19(14)
C(1)-C(5)-C(7)-O(2)
-109.81(14)
C(6)-C(5)-C(7)-O(2)
9.18(17)
N(1)-C(5)-C(7)-O(3)
-52.92(14)
C(1)-C(5)-C(7)-O(3)
67.08(13)
C(6)-C(5)-C(7)-O(3)
-173.94(10)
C(5)-N(1)-C(9)-O(4)
2.4(2)
C(5)-N(1)-C(9)-C(10) -177.45(11)
- 152 -
X-ray data
Table 7
Hydrogen bonds for hs1 [pm and °]
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
N(1)-H(1)...O(1)
82.9(18)
227.3(17)
267.31(19)
110.0(14)
N(1)-H(1)...O(5)
82.9(18)
225.1(17)
264.82(19)
109.7(14)
- 153 -
X-ray data
Tetraethyl but-3-yne-1,1-diyldiphosphonate (34)
Table 1
Crystal data and structure refinement for 34
Identification code
hs2
Empirical formula
C15 H26 O6 P2
Formula weight
364.30
Temperature
173(2) K
Wavelength
71.073 pm
Crystal system
Monoclinic
Space group
P2(1)
Unit cell dimensions
a = 916.70(10) pm
Į = 90°
b = 2503.6(3) pm
ȕ = 117.550(10)°
c = 939.20(10) pm
Ȗ = 90°
Volume
1.9111(4) nm3
Z
4
Density (calculated)
1.266 Mg/m3
Absorption coefficient
0.252 mm-1
- 154 -
X-ray data
F(000)
776
Crystal size
1.0 x 0.7 x 0.4 mm3
Theta range for data collection
2.57 to 27.50°.
Reflections collected
5475
Independent reflections
4352 [R(int) = 0.0503]
Completeness to theta = 27.50°
99.0 %
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
4352 / 0 / 212
Goodness-of-fit on F2
1.026
Final R indices [I>2sigma(I)]
R1 = 0.0713, wR2 = 0.1994
R indices (all data)
R1 = 0.0815, wR2 = 0.2105
Extinction coefficient
0.0035(17)
Largest diff. peak and hole
1.567 and -1.278 e.Å-3
Table 2
Atomic coordinates (x 104) and equivalent isotropic displacement parameters (pm2x
10-1) for hs2. U(eq) is defined as one third of the trace of the orthogonalized Uij
tensor
x
y
z
U(eq)
P(1)
9096(1)
1398(1)
4496(1)
24(1)
P(2)
6860(1)
939(1)
5752(1)
27(1)
O(1)
8002(3)
1613(1)
2899(3)
34(1)
O(2)
10869(3)
1652(1)
5276(3)
36(1)
O(3)
9461(3)
783(1)
4609(3)
33(1)
O(4)
7575(3)
416(1)
6403(4)
44(1)
O(5)
5676(3)
977(1)
3898(3)
35(1)
O(6)
5785(3)
1173(1)
6524(3)
34(1)
C(1)
9077(5)
1539(2)
10089(5)
52(1)
- 155 -
X-ray data
Table 3
C(2)
9422(4)
1479(2)
9022(4)
38(1)
C(3)
9851(4)
1412(1)
7709(4)
31(1)
C(4)
8348(3)
1468(1)
6000(3)
22(1)
C(5)
7482(3)
2020(1)
5773(4)
25(1)
C(6)
8588(4)
2476(1)
6077(4)
31(1)
C(7)
9503(5)
2842(1)
6390(5)
42(1)
C(8)
11316(5)
2136(2)
4681(7)
59(1)
C(9)
12506(7)
2035(2)
4130(8)
76(2)
C(10)
10030(5)
531(2)
3554(5)
49(1)
C(11)
8680(8)
242(2)
2243(6)
77(2)
C(12)
4112(6)
691(2)
3250(6)
59(1)
C(13)
3347(6)
678(2)
1507(6)
66(1)
C(14)
5543(7)
883(2)
7746(7)
65(1)
C(15)
4467(12)
984(7)
7943(15)
460(20)
Bond lengths [pm] and angles [°] for 34
P(1)-O(1)
146.7(2)
P(1)-O(3)
156.9(2)
P(1)-O(2)
157.6(2)
P(1)-C(4)
184.2(3)
P(2)-O(4)
146.6(2)
P(2)-O(5)
157.2(3)
P(2)-O(6)
158.2(2)
P(2)-C(4)
183.7(3)
O(2)-C(8)
146.9(4)
O(3)-C(10)
145.9(4)
O(5)-C(12)
145.9(4)
O(6)-C(14)
145.8(4)
C(1)-C(2)
119.2(6)
C(2)-C(3)
146.7(4)
C(3)-C(4)
156.5(4)
C(4)-C(5)
155.8(4)
- 156 -
X-ray data
C(5)-C(6)
146.5(4)
C(6)-C(7)
118.4(5)
C(8)-C(9)
143.0(6)
C(10)-C(11)
147.0(7)
C(12)-C(13)
145.3(6)
C(14)-C(15)
111.3(8)
O(1)-P(1)-O(3)
116.97(14)
O(1)-P(1)-O(2)
113.38(13)
O(3)-P(1)-O(2)
102.92(13)
O(1)-P(1)-C(4)
115.33(12)
O(3)-P(1)-C(4)
100.66(12)
O(2)-P(1)-C(4)
105.93(13)
O(4)-P(2)-O(5)
117.52(16)
O(4)-P(2)-O(6)
113.16(14)
O(5)-P(2)-O(6)
103.18(13)
O(4)-P(2)-C(4)
115.39(14)
O(5)-P(2)-C(4)
100.89(12)
O(6)-P(2)-C(4)
104.99(13)
C(8)-O(2)-P(1)
124.5(2)
C(10)-O(3)-P(1)
120.6(2)
C(12)-O(5)-P(2)
118.3(2)
C(14)-O(6)-P(2)
122.0(2)
C(1)-C(2)-C(3)
179.4(4)
C(2)-C(3)-C(4)
113.6(2)
C(5)-C(4)-C(3)
111.7(2)
C(5)-C(4)-P(2)
108.70(18)
C(3)-C(4)-P(2)
108.42(19)
C(5)-C(4)-P(1)
109.05(18)
C(3)-C(4)-P(1)
108.33(18)
P(2)-C(4)-P(1)
110.61(14)
C(6)-C(5)-C(4)
113.7(2)
C(7)-C(6)-C(5)
177.2(4)
C(9)-C(8)-O(2)
112.7(4)
O(3)-C(10)-C(11) 110.8(4)
C(13)-C(12)-O(5) 110.5(4)
C(15)-C(14)-O(6) 119.2(6)
- 157 -
X-ray data
Table 4
Anisotropic displacement parameters (pm2x 10-1) for hs2. The anisotropic
displacement factor exponent takes the form: -2 2[h2 a*2U11 + ... + 2 h k a* b* U12]
U11
U22
U33
U23
U13
U12
P(1)
22(1)
29(1)
26(1)
4(1)
16(1)
4(1)
P(2)
27(1)
23(1)
39(1)
4(1)
22(1)
1(1)
O(1)
32(1)
45(1)
29(1)
7(1)
18(1)
9(1)
O(2)
24(1)
46(1)
43(1)
11(1)
19(1)
-1(1)
O(3)
41(1)
31(1)
38(1)
1(1)
28(1)
9(1)
O(4)
46(1)
26(1)
73(2)
13(1)
40(1)
7(1)
O(5)
33(1)
35(1)
39(1)
-6(1)
19(1)
-13(1)
O(6)
33(1)
38(1)
47(1)
11(1)
30(1)
7(1)
C(2)
35(2)
51(2)
26(1)
3(1)
13(1)
7(1)
C(3)
26(1)
43(2)
26(1)
3(1)
13(1)
4(1)
C(4)
21(1)
25(1)
25(1)
2(1)
14(1)
2(1)
C(5)
24(1)
23(1)
31(1)
-3(1)
14(1)
1(1)
C(6)
32(2)
29(1)
32(2)
-2(1)
15(1)
0(1)
C(7 )
45(2)
33(2)
47(2)
-7(1)
22(2)
-8(1)
C(8)
42(2)
58(3)
87(3)
28(2)
38(2)
-3(2)
C(9)
68(3)
78(3)
107(4)
32(3)
61(3)
8(3)
C(10)
62(2)
49(2)
51(2)
0(2)
40(2)
23(2)
C(11)
110(4)
61(3)
44(2)
-10(2)
22(3)
21(3)
C(12)
51(2)
66(3)
60(3)
-19(2)
26(2)
-34(2)
C(13)
55(3)
64(3)
54(2)
-2(2)
5(2)
-20(2)
C(14)
76(3)
70(3)
81(3)
35(3)
65(3)
16(2)
C(15)
201(11)
940(50)
390(20)
560(30)
261(15)
370(2)
- 158 -
X-ray data
Table 5
Hydrogen coordinates (x 104) and isotropic displacement parameters (pm2x 10 -1)
for hs2
x
y
z
U(eq)
H(3A)
10352
1055
7802
83(5)
H(3B)
10688
1683
7826
83(5)
H(5A)
6580
2044
4660
83(5)
H(5B)
6984
2041
6508
83(5)
H(8A)
10313
2288
3789
83(5)
H(8B)
11763
2404
5553
83(5)
H(9A)
13527
1906
5026
99(6)
H(9B)
12726
2366
3706
99(6)
H(9C)
12079
1764
3280
99(6)
H(10A)
10933
279
4182
83(5)
H(10B)
10466
808
3099
83(5)
H(11A)
8234
-26
2695
99(6)
H(11B)
9096
66
1572
99(6)
H(11C)
7811
495
1589
99(6)
H(12A)
4300
322
3674
83(5)
H(12B)
3367
871
3597
83(5)
H(13A)
3104
1043
1086
99(6)
H(13B)
2323
472
1094
99(6)
H(13C)
4099
509
1165
99(6)
H(14A)
5471
499
7480
83(5)
H(14B)
6547
933
8782
83(5)
H(15A)
4679
1320
8542
99(6)
H(15B)
4290
697
8556
99(6)
H(15C)
3485
1023
6903
99(6)
H(1)
8642
1602
11199
110(20)
H(7)
10200
3130
6666
54(12)
- 159 -
X-ray data
Tetraethyl 2-(1-benzyl-1H-1,2,3-triazol-4-yl)ethane-1,1-diyldiphosphonate (37)
Table 1
Crystal data and structure refinement for 37
Identification code
hs4
Empirical formula
C29 H40 N6 O6 P2
Formula weight
630.61
Temperature
173(2) K
Wavelength
71.073 pm
Crystal system
Triclinic
Space group
P -1
Unit cell dimensions
a = 861.5(6) pm
Į = 88.06(3)°
b = 1282.9(4) pm
ȕ = 78.28(4)°
c = 1499.6(5) pm
Ȗ = 75.70(4)°
Volume
1.5723(13) nm3
Z
2
Density (calculated)
1.332 Mg/m3
Absorption coefficient
0.190 mm-1
- 160 -
X-ray data
F(000)
668
Crystal size
0.9 x 0.2 x 0.2 mm3
Theta range for data collection
2.60 to 25.00°.
Reflections collected
6834
Independent reflections
5524 [R(int) = 0.0273]
Completeness to theta = 25.00°
99.5 %
Absorption correction
None
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
5524 / 45 / 428
Goodness-of-fit on F2
1.024
Final R indices [I>2sigma(I)]
R1 = 0.0702, wR2 = 0.1681
R indices (all data)
R1 = 0.1135, wR2 = 0.1940
Extinction coefficient
0.007(2)
Largest diff. peak and hole
0.617 and -0.739 e.Å-3
Table 2
Atomic coordinates (x 104) and equivalent isotropic displacement parameters (pm2x
10-1) for hs4 U(eq) is defined as on third of the trace of the orthogonalized Uij tensor.
x
y
z
U(eq)
P(1)
1577(1)
605(1)
3447(1)
29(1)
O(1)
1878(4)
480(2)
4372(2)
39(1)
O(2)
-166(3)
1285(2)
3367(2)
35(1)
C(1)
-1516(6)
1461(5)
4101(4)
95(3)
C(2)
-2793(5)
2407(4)
3975(4)
57(1)
O(3)
1762(4)
-495(2)
2935(2)
39(1)
C(3)
799(8)
-1182(5)
3375(5)
33(2)
C(4)
1791(13)
-2271(4)
3467(6)
44(3)
C(30)
2113(9)
-1525(5)
3311(6)
39(2)
-1919(8)
3615(8)
60(3)
C(40)
637(14)
- 161 -
X-ray data
C(9)
2927(5)
1290(3)
2667(3)
25(1)
P(2)
4921(1)
347(1)
2271(1)
32(1)
O(4)
5016(4)
-310(2)
1480(2)
46(1)
O(5)
6121(4)
1102(3)
2130(2)
49(1)
C(5)
7609(6)
971(4)
1491(3)
54(1)
C(6)
8074(7)
1992(4)
1319(3)
65(2)
O(6)
5285(6)
-309(4)
3104(3)
95(2)
C(7)
6085(13)
-1341(5)
3307(5)
50(3)
C(70)
6136(16)
-546(9)
3810(7)
121(8)
C(8)
6120(40)
-1472(11)
4277(9)
81(5)
C(80)
6470(40)
-1683(10)
4043(13)
81(5)
C(10)
3170(5)
2249(3)
3204(3)
27(1)
C(11)
1651(5)
3119(3)
3540(3)
27(1)
N(1)
854(5)
3197(3)
4428(2)
35(1)
N(2)
-367(5)
4069(3)
4538(2)
39(1)
N(3)
-320(4)
4551(3)
3724(2)
31(1)
C(12)
917(5)
3978(3)
3097(3)
30(1)
C(13)
-1499(5)
5551(3)
3615(3)
40(1)
C(14)
-2904(5)
5352(3)
3248(3)
34(1)
C(15)
-2695(6)
5034(4)
2345(3)
41(1)
C(16)
-3944(7)
4806(4)
2007(4)
51(1)
C(17)
-5433(7)
4881(4)
2563(4)
54(1)
C(18)
-5695(6)
5206(5)
3446(4)
61(2)
C(19)
-4443(6)
5448(4)
3798(3)
49(1)
C(20)
2160(5)
1667(3)
1810(3)
29(1)
C(21)
3152(5)
2191(3)
1074(3)
27(1)
N(4)
3080(5)
3263(3)
1101(2)
35(1)
N(5)
4019(5)
3512(3)
353(2)
36(1)
N(6)
4678(4)
2599(2)
-142(2)
29(1)
C(22)
4172(5)
1772(3)
278(3)
29(1)
C(23)
5789(5)
2600(3)
-1036(3)
32(1)
C(24)
7421(5)
2787(3)
-950(2)
29(1)
C(25)
7561(5)
3796(3)
-737(3)
33(1)
C(26)
9058(6)
3964(4)
-671(3)
45(1)
C(27)
10440(6)
3122(4)
-813(3)
49(1)
C(28)
10315(6)
2111(4)
-1026(3)
48(1)
C(29)
8822(6)
1944(4)
-1090(3)
40(1)
- 162 -
X-ray data
Table 3
Bond lengths [pm] and angles [°] for hs4
P(1)-O(1)
145.8(3)
C(17)-C(18)
135.8(8)
P(1)-O(2)
156.5(3)
C(18)-C(19)
139.5(7)
P(1)-O(3)
158.8(3)
C(20)-C(21)
150.0(5)
P(1)-C(9)
183.7(4)
C(21)-N(4)
136.3(5)
O(2)-C(1)
140.9(4)
C(21)-C(22)
136.7(5)
C(1)-C(2)
145.9(5)
N(4)-N(5)
132.3(5)
O(3)-C(30)
140.6(5)
N(5)-N(6)
134.3(4)
O(3)-C(3)
141.3(4)
N(6)-C(22)
133.6(5)
C(3)-C(4)
146.5(6)
N(6)-C(23)
148.1(5)
C(30)-C(40)
146.5(6)
C(23)-C(24)
151.5(6)
C(9)-C(20)
157.1(5)
C(24)-C(25)
138.3(6)
C(9)-C(10)
157.2(5)
C(24)-C(29)
139.1(6)
C(9)-P(2)
183.7(4)
C(25)-C(26)
138.2(6)
P(2)-O(4)
145.6(3)
C(26)-C(27)
138.0(7)
P(2)-O(6)
152.4(4)
C(27)-C(28)
138.1(7)
P(2)-O(5)
156.4(3)
C(28)-C(29)
137.8(7)
O(5)-C(5)
141.3(4)
O(1)-P(1)-O(2)
115.14(17)
C(5)-C(6)
146.1(5)
O(1)-P(1)-O(3)
114.30(17)
O(6)-C(7)
138.8(5)
O(2)-P(1)-O(3)
104.07(17)
O(6)-C(70)
139.1(5)
O(1)-P(1)-C(9)
115.13(18)
C(7)-C(8)
146.4(6)
O(2)-P(1)-C(9)
102.54(17)
C(70)-C(80)
145.9(6)
O(3)-P(1)-C(9)
104.16(16)
C(10)-C(11)
150.3(5)
C(1)-O(2)-P(1)
123.0(3)
C(11)-C(12)
135.4(6)
O(2)-C(1)-C(2)
112.3(3)
C(11)-N(1)
136.3(5)
C(30)-O(3)-C(3)
45.6(4)
N(1)-N(2)
132.0(5)
C(30)-O(3)-P(1)
125.6(4)
N(2)-N(3)
134.8(5)
C(3)-O(3)-P(1)
116.5(4)
N(3)-C(12)
134.6(5)
O(3)-C(3)-C(4)
111.8(4)
N(3)-C(13)
145.4(5)
O(3)-C(30)-C(40) 112.0(4)
C(13)-C(14)
150.5(6)
C(20)-C(9)-C(10) 112.8(3)
C(14)-C(19)
139.1(6)
C(20)-C(9)-P(1)
109.3(3)
C(14)-C(15)
139.1(6)
C(10)-C(9)-P(1)
108.1(3)
C(15)-C(16)
137.3(7)
C(20)-C(9)-P(2)
107.7(3)
C(16)-C(17)
136.4(8)
C(10)-C(9)-P(2)
109.2(3)
- 163 -
X-ray data
P(1)-C(9)-P(2)
109.68(19)
N(4)-N(5)-N(6)
106.8(3)
O(4)-P(2)-O(6)
113.5(3)
C(22)-N(6)-N(5)
111.1(3)
O(4)-P(2)-O(5)
115.00(18)
C(22)-N(6)-C(23)
128.2(3)
O(6)-P(2)-O(5)
104.6(3)
N(5)-N(6)-C(23)
120.7(3)
O(4)-P(2)-C(9)
114.86(19)
N(6)-C(22)-C(21)
105.5(3)
O(6)-P(2)-C(9)
105.1(2)
N(6)-C(23)-C(24)
112.6(3)
O(5)-P(2)-C(9)
102.50(18)
C(25)-C(24)-C(29)
118.4(4)
C(5)-O(5)-P(2)
127.4(3)
C(25)-C(24)-C(23)
121.1(4)
O(5)-C(5)-C(6)
111.4(3)
C(29)-C(24)-C(23)
120.6(4)
C(7)-O(6)-C(70)
55.9(6)
C(26)-C(25)-C(24)
120.8(4)
C(7)-O(6)-P(2)
138.9(5)
C(27)-C(26)-C(25)
120.3(4)
C(70)-O(6)-P(2)
150.3(7)
C(26)-C(27)-C(28)
119.4(5)
O(6)-C(7)-C(8)
112.7(5)
C(29)-C(28)-C(27)
120.2(5)
O(6)-C(70)-C(80)
113.1(5)
C(28)-C(29)-C(24)
120.9(4)
C(11)-C(10)-C(9)
115.7(3)
C(12)-C(11)-N(1)
108.1(4)
C(12)-C(11)-C(10)
129.6(4)
N(1)-C(11)-C(10)
122.1(3)
N(2)-N(1)-C(11)
108.9(3)
N(1)-N(2)-N(3)
106.9(3)
C(12)-N(3)-N(2)
110.4(3)
C(12)-N(3)-C(13)
128.8(4)
N(2)-N(3)-C(13)
120.8(3)
N(3)-C(12)-C(11)
105.8(4)
N(3)-C(13)-C(14)
111.6(3)
C(19)-C(14)-C(15)
117.3(4)
C(19)-C(14)-C(13)
121.8(4)
C(15)-C(14)-C(13)
120.9(4)
C(16)-C(15)-C(14)
121.8(5)
C(17)-C(16)-C(15)
120.0(5)
C(18)-C(17)-C(16)
120.1(5)
C(17)-C(18)-C(19)
120.7(5)
C(14)-C(19)-C(18)
120.2(5)
C(21)-C(20)-C(9)
116.4(3)
N(4)-C(21)-C(22)
107.7(3)
N(4)-C(21)-C(20)
121.8(3)
C(22)-C(21)-C(20)
130.4(4)
N(5)-N(4)-C(21)
108.9(3)
- 164 -
X-ray data
Table 4
Anisotropic displacement parameters (pm2x 10-1) for hs4.
displacement factor exponent takes the form: -2
2[
The anisotropic
h2 a*2U11 + ... + 2 h k a* b* U12 ]
U11
U22
U33
U23
U13
P(1)
34(1)
28(1)
27(1)
4(1)
-1(1)
-17(1)
O(1)
47(2)
39(2)
33(2)
11(1)
-8(1)
-21(2)
O(2)
31(2)
45(2)
28(2)
0(1)
1(1)
-15(1)
C(1)
36(3)
136(6)
82(4)
68(4)
24(3)
-1(4)
C(2)
34(3)
72(4)
64(3)
-18(3)
1(3)
-18(3)
O(3)
56(2)
28(2)
37(2)
-1(1)
4(2)
-27(2)
C(3)
50(6)
30(5)
27(4)
2(4)
3(4)
-32(5)
C(4)
73(8)
30(5)
29(5)
5(4)
9(5)
-28(5)
C(30)
39(6)
27(5)
51(6)
1(4)
-3(5)
-11(4)
C(40)
60(8)
78(9)
48(7)
-2(6)
8(6)
-42(7)
C(9)
26(2)
22(2)
27(2)
2(2)
-2(2)
-11(2)
P(2)
33(1)
26(1)
36(1)
4(1)
0(1)
-9(1)
O(4)
45(2)
30(2)
58(2)
-13(1)
6(2)
-14(2)
O(5)
35(2)
58(2)
53(2)
-24(2)
17(2)
-26(2)
C(5)
50(3)
55(3)
51(3)
-13(2)
17(2)
-20(3)
C(6)
73(4)
95(4)
44(3)
7(3)
-7(3)
-55(4)
O(6)
69(3)
110(4)
74(3)
51(3)
-1(2)
21(3)
C(7)
64(7)
32(5)
39(5)
1(4)
-6(5)
13(5)
C(70)
72(10)
75(10)
195(19)
-79(12)
47(12)
-31(9)
C(8)
152(15)
27(6)
82(9)
6(6)
-74(11)
-13(7)
C(80)
152(15)
27(6)
82(9)
6(6)
-74(11)
-13(7)
C(10)
31(2)
24(2)
29(2)
0(2)
-7(2)
-13(2)
C(11)
26(2)
28(2)
31(2)
-1(2)
-7(2)
-13(2)
N(1)
42(2)
32(2)
34(2)
-3(2)
-4(2)
-14(2)
N(2)
41(2)
38(2)
40(2)
-6(2)
-4(2)
-15(2)
N(3)
29(2)
32(2)
34(2)
-5(2)
-6(2)
-11(2)
C(12)
29(2)
32(2)
32(2)
-1(2)
-4(2)
-14(2)
C(13)
34(3)
30(2)
55(3)
-9(2)
-10(2)
-6(2)
C(14)
29(2)
26(2)
46(3)
-2(2)
-3(2)
-9(2)
C(15)
38(3)
43(3)
42(3)
3(2)
-3(2)
-14(2)
- 165 -
U12
X-ray data
C(16)
63(4)
50(3)
50(3)
8(2)
-25(3)
-20(3)
C(17)
50(3)
55(3)
72(4)
18(3)
-31(3)
-27(3)
C(18)
33(3)
73(4)
76(4)
20(3)
-3(3)
-20(3)
C(19)
41(3)
53(3)
48(3)
1(2)
1(2)
-9(2)
C(20)
31(2)
29(2)
28(2)
4(2)
-2(2)
-12(2)
C(21)
27(2)
24(2)
28(2)
1(2)
-2(2)
-10(2)
N(4)
45(2)
23(2)
34(2)
0(1)
3(2)
-11(2)
N(5)
49(2)
23(2)
32(2)
-1(1)
8(2)
-12(2)
N(6)
38(2)
24(2)
25(2)
0(1)
1(2)
-13(2)
C(22)
39(3)
20(2)
31(2)
1(2)
-4(2)
-15(2)
C(23)
39(3)
30(2)
26(2)
1(2)
4(2)
-13(2)
C(24)
33(2)
34(2)
19(2)
2(2)
2(2)
-14(2)
C(25)
35(3)
27(2)
38(2)
2(2)
-5(2)
-13(2)
C(26)
47(3)
41(3)
53(3)
4(2)
-11(2)
-22(2)
C(27)
36(3)
65(3)
49(3)
8(2)
-6(2)
-20(3)
C(28)
31(3)
53(3)
49(3)
0(2)
1(2)
1(2)
C(29)
45(3)
34(2)
35(2)
-1(2)
6(2)
-10(2)
_________________________________________________________________________________
Table 5
Hydrogen coordinates (x 104) and isotropic displacement parameters (pm2x 10 -1) for
hs4
x
y
z
U(eq)
H(1A)
-1145
1554
4669
57(4)
H(1B)
-1975
821
4168
57(4)
H(2A)
-2321
3030
3847
91(7)
H(2B)
-3645
2548
4529
91(7)
H(2C)
-3273
2277
3462
91(7)
H(3A)
-40
-1216
3021
57(4)
H(3B)
228
-880
3987
57(4)
H(4A)
2537
-2251
3877
91(7)
H(4B)
2426
-2550
2868
91(7)
- 166 -
X-ray data
H(4C)
1073
-2740
3719
91(7)
H(30A)
2642
-1501
3834
57(4)
H(30B)
2892
-2033
2849
57(4)
H(40A)
-145
-1412
4065
91(7)
H(40B)
927
-2625
3893
91(7)
H(40C)
143
-1984
3092
91(7)
H(5A)
7494
677
913
57(4)
H(5B)
8483
449
1724
57(4)
H(6A)
7240
2497
1056
91(7)
H(6B)
9129
1870
892
91(7)
H(6C)
8166
2292
1893
91(7)
H(7A)
7222
-1507
2953
57(4)
H(7B)
5533
-1862
3117
57(4)
H(70A)
5492
-114
4356
57(4)
H(70B)
7185
-334
3633
57(4)
H(8A)
5163
-970
4635
91(7)
H(8B)
7119
-1322
4395
91(7)
H(8C)
6089
-2211
4451
91(7)
H(80A)
5452
-1920
4149
91(7)
H(80B)
6929
-1785
4595
91(7)
H(80C)
7256
-2106
3540
91(7)
H(10A)
3647
1955
3734
57(4)
H(10B)
3975
2580
2803
57(4)
H(12)
1213
4142
2473
51(4)
H(13A)
-948
6019
3192
57(4)
H(13B)
-1923
5931
4211
57(4)
H(15)
-1661
4971
1952
51(4)
H(16)
-3771
4597
1386
51(4)
H(17)
-6289
4706
2333
51(4)
H(18)
-6740
5270
3826
51(4)
H(19)
-4642
5678
4415
51(4)
H(20A)
1954
1032
1540
57(4)
H(20B)
1087
2179
2018
57(4)
H(22)
4460
1046
68
51(4)
H(23A)
5258
3170
-1415
57(4)
H(23B)
5976
1900
-1350
57(4)
H(25)
6617
4380
-634
51(4)
H(26)
9137
4662
-527
51(4)
- 167 -
X-ray data
Table 6
H(27)
11467
3238
-765
51(4)
H(28)
11261
1529
-1128
51(4)
H(29)
8747
1245
-1233
51(4)
Torsion angles [°] for hs4
______________________________________________________________________________
O(1)-P(1)-O(2)-C(1)
-20.6(5)
O(3)-P(1)-O(2)-C(1)
105.3(5)
C(9)-P(1)-O(2)-C(1)
-146.4(5)
P(1)-O(2)-C(1)-C(2)
158.5(4)
O(1)-P(1)-O(3)-C(30)
2.6(5)
O(2)-P(1)-O(3)-C(30) -123.8(5)
C(9)-P(1)-O(3)-C(30) 129.1(4)
O(1)-P(1)-O(3)-C(3)
55.5(5)
O(2)-P(1)-O(3)-C(3)
-70.9(4)
C(9)-P(1)-O(3)-C(3)
-178.0(4)
C(30)-O(3)-C(3)-C(4) -11.9(7)
P(1)-O(3)-C(3)-C(4)
-127.0(6)
C(3)-O(3)-C(30)-C(40) 5.4(7)
P(1)-O(3)-C(30)-C(40) 99.2(8)
O(1)-P(1)-C(9)-C(20) -163.5(2)
O(2)-P(1)-C(9)-C(20) -37.7(3)
O(3)-P(1)-C(9)-C(20)
70.5(3)
O(1)-P(1)-C(9)-C(10) -40.4(3)
O(2)-P(1)-C(9)-C(10)
85.4(3)
O(3)-P(1)-C(9)-C(10) -166.4(3)
O(1)-P(1)-C(9)-P(2)
78.6(2)
O(2)-P(1)-C(9)-P(2) -155.62(19)
O(3)-P(1)-C(9)-P(2)
-47.4(2)
C(20)-C(9)-P(2)-O(4) -32.9(3)
C(10)-C(9)-P(2)-O(4) -155.7(2)
P(1)-C(9)-P(2)-O(4)
86.0(2)
C(20)-C(9)-P(2)-O(6) -158.4(3)
C(10)-C(9)-P(2)-O(6)
78.8(3)
- 168 -
X-ray data
P(1)-C(9)-P(2)-O(6)
-39.5(3)
C(20)-C(9)-P(2)-O(5)
92.6(3)
C(10)-C(9)-P(2)-O(5) -30.3(3)
P(1)-C(9)-P(2)-O(5)
-148.6(2)
O(4)-P(2)-O(5)-C(5)
-24.1(5)
O(6)-P(2)-O(5)-C(5)
101.1(5)
C(9)-P(2)-O(5)-C(5)
-149.4(4)
P(2)-O(5)-C(5)-C(6)
158.5(4)
O(4)-P(2)-O(6)-C(7)
21.8(10)
O(5)-P(2)-O(6)-C(7) -104.3(10)
C(9)-P(2)-O(6)-C(7)
148.1(10)
O(4)-P(2)-O(6)-C(70) 128.6(13)
O(5)-P(2)-O(6)-C(70)
2.5(13)
- 169 -
Summary
Chapter 6
Summary
In recent years, we have witnessed a huge progress in developing new drugs.
However, the process of their design is expensive and long. Finding an effective
synthetic pathway which can be used to synthesize new drugs plays a crucial role
here. Undoubtedly, inhibitors of matrix metalloproteinase are very attractive
compounds for drug discovery. According to much excisting evidence, they play
a fundamental role in a wide variety of pathologies. The implementation of MMPs into
processes critical to angiogenesis, tumor invasion etc., has prompted rapid
development of new important agents, namely inhibitors of matrix metalloporteinase.
In this thesis, different and effective strategies that can be employed to design
potential inhibitors against overexpression of matrix metalloproteinases have been
demonstrated. Moreover, strategies that can highlight the strength and drawback of
each approach have been investigated. We have synthesied a family of different
compounds bearing in their molecule oxalyl, phosphonoformyl and bisphosphonate
groups’ theoretically capable to chelate to metal ions like zinc, calcium or magnesium.
These agents are referred to as a potent zinc binding groups (ZBGs). It should be
noted that, the past few decades are testament to the ingenuity of chemistry in
designing such chemical entries.
First of all, convenient synthetic routes to methyl 2-oxalylimino and 2(phosphonformimido)-3,3,3-trifluoropropanoates have been elaborated, based on the
reaction
of
methyl
trifluoropyruvate
with
ethyl
oxamate
or
diethyl
carbamoylphosphonate, respectively followed by dehydratation using dehydrating
agent [Scheme 43].
- 170 -
Summary
O
CF3
1.
H2N
X
O
O
O
O
CF3
O
N
O
O
2.
O
TFAA/Py
O
X
O
X = C, P-OEt
Scheme 43
General synthetic route to methyl 2-oxalylimino and 2-(phosphonoformimido)-3,3,3trifluoropropanoates
Methyl trifluoropyruvate used as a fluorine containig ketone for the condensation
reaction with both imines allowed introducing to the molecule of imines trifluoromethyl
group. The presence of the Tfm group can not only exert considerable polarization
effects on the neighbouring substituents by also influence therapeutic index. By
examining the nature and reactivity of the new imines we found that they are powerful
ʌ-donor
electrophilies to be alkylated by different organometallic reagents and
heteroaromatic compounds [Scheme 44].
CF3
O
F3C
O
N
O
X
O
R
O
O
O
O
N
X
H
O
O
X = C, P-OEt
Scheme 44
General synthetic route Į-Tfm-Į-amino acids derivatives
To be more precise, the compounds obtained by addition of Grignard reagents (R1)
and ʌ-donors aromatic compounds (R2) to imines possessing on the nitrogen atom Noxalyl and N-phosphonoformyl groups [Figure 37], are useful synthetic intermediates
towards a variety of 3,3,3-trifluoroalanine derivatives-potential drug candidates [Figure
37].
- 171 -
Summary
F3 C
R1
F3C
O
O
X
X
MeO2C
R2
N
H
MeO2C
N
H
O
O
X = C, P-OEt
X = C, P-OEt
NH2
;
R2 =
R1 = CH3, Ph, Bz, Allyl
O
;
N
CH3
Figure 37
H 3C
;
;
N
H
N
etc.
O
Ph
3,3,3-trifluoroalanine derivatives bearing on the nitrogen atom N-oxalyl and Nphosphonoformyl potential ZBGs groups
The presented N-oxalyl and N-phosphonoformyl derivatives of Į-Tfm-Į-amino acids
are not only potential targets in treatment of much pathology connected with
overexpression of humans’ matrix metalloproteinases. In addition, the novel fluorinated
amino acid derivatives demonstrated here could find further application as building
blocks for the modification of other biologically active peptides. The implementation of
these molecules into unnatural proteins can pave the way for the de novo design and
application. Obviously, fluor modification of N-oxalyl and N-phosphonoformyl
derivatives of Į-Tfm-Į-amino acids, can serve as a “final push” towards higher activity
and stability after rational design.
Secondly, an efficient synthetic approach giving the possibility for facile and
rapid synthesis of a novel nitrogen containing bisphosphonates (N-BPs) based on the
“click methodology” has been demonstrated. Recently, it has been an ongoing
challenge to develop highly efficient strategies for the design of different derivatives of
N-BPs because they have the potential to become one of the most popular matrix
metalloproteinase
inhibitors.
The
Cu(I)
catalyzed
1,3-cycloaddition
between
monopropargyl bisphosphonate and different azides was shown to give access to
a wide variety of 1,4-disubstituted triazoles [Scheme 45].
- 172 -
Summary
O
O
P
R
N3
P
P
OEt
Cu(I)
OEt
OEt
P
N
OEt
R
N
OEt
OEt
OEt
OEt
O
N
O
O
NH
N
AcO
O
R = Ph, Bz, C2H4C6F13,
O
AcO
AcO
O
O
OH
OAc
O
Scheme 45 A new class of nitrogen containing bisphosphonates (N-BPs)
It was found that not only benzyl azide or phenyl azide are ideal precursors for the
synthesis of modified bisphosphonates but also highly functionalized azides like
2,3,4,5-tetra-O-acetyl-ȕ-D-glucopyranosyl azide or 3’-azido-3’deoxythymidine (AZT).
The aza analogue of Zolendronate (the most potent bisphosphonate to date) - a free
N-bisphosphonate was successfully synthesized from N-pivaloyl methyl derivative
[Figure 38].
O
P
O
OH
P
OH
N
HO
P
OH
P
N
OH
N
OH
OH
H
O
N
N
OH
OH
O
Zolendronate
2-(1H-1,2,3-Triazol-4-yl)ethane-1,1,-diyl-bis(phosphonic acid)
(1-hydroxy-2-imidazol-1-yl)ethane-1,1,-diyl-bis(phosphonic acid)
Figure 38 Aza-analogue of Zolendronic acid
It is important to add that a new method for the preparation of monopropargyl
bisphosphonate based on the selective addition of sodium acetylide to vinylidene
- 173 -
Summary
bisphosphonate with high yield was presented [Scheme 46]. Monopropargyl
bisphosphonate is very interesting counterpart for the application to the click
chemistry.
O
O
OEt
OEt
P
OEt
OEt
o
THF/ -20 C
OEt
P
P
Na
OEt
P
OEt
OEt
O
O
Scheme 46 Synthetic pathway to monopropargyl bisphosphonate
Furthermore, the synthesis of the bisphosphonates containing two triazole moieties via
the cycloaddition reaction between bis-propargyl-substituted bisphosphonate and two
equivalents of azides has been demonstrated [Scheme 47].
O
EtO
EtO
OEt
P
2 R-N 3
OEt
O
O
P
P
OEt
OEt
Cu(I)
+
P
OEt
N
OEt
R
O
N
N
N
N
N
R
OAc
R=
Bz
;
C 2H 4C 6 F13
; AcO
AcO
O
OAc
Scheme 47 N-Bisphosphonate molecules with ditriazoles moieties
Next, the method allowing for ligation in situ two nitrogen-containing BPs to one
another via one pot reaction [Scheme 48] has been presented.
- 174 -
Summary
R
NaN3 ,
O
P
P
R
OEt
OEt
Br
N
N
Br
N
OEt
OEt
N
R
R
x NaBr
N
N
Cu(I)
O
(EtO)2 P
O
(EtO)2 P
P(OEt) 2
P(OEt) 2
O
O
O
R = H, F
Scheme 48 N-Bisphosphonates molecules with ditriazoles moieties via one pot reaction
Finally, we have shown an effective strategy that can be employed to design
fluorinated bisphosphonates via Arbuzov/Perkow. We found that phosphite esters
react
easily
with
2-chloro-2,2,-difluoroacetyl
chloride
yielding
vinylidene
bisphosphonates difluoro (phosphonooxy)vinyliphosphonate. Taking into account the
fact that difluoromethylidene bisphosphonates (F2MBP) were found to inhibit bone
resorption both in vivo and in vitro, we believe that new fluorinated derivatives of BPs
are capable to become useful inhibitors of MMPs [Figure 39].
O
O
F2C
P
C
P
OR
OR
OR
OR
O
R= SiMe3, Et)
Figure 39 Difluoromethylidene derivative containing phosphonooxy and phosphonate groups
Most of the compounds presented in this dissertation will be examined for
matrix metalloproteinases inhibitor activity. We believe that the presented compounds
can find application as drug candidates*. Further studies are under development in our
laboratories.
*
tumours,
osteoporosis,
and
other
bone
- 175 -
related
diseases,
Herpex,
AIDS,
etc.
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27
- 186 -
List of compounds
Chapter 8
List of compounds
O
F3C
F
F
F
OMe
F3C
C
O
O
1
2
O
O
OEt
OEt
H2N
H2N
P
O
O
4
3
F3C
OH
OEt
O
F3C
OH
O
OEt
MeO2C
OEt
N
H
MeO2C
N
H
O
P
OEt
O
5
6
- 187 -
List of compounds
CF3
CF3
O
O
OEt
OEt
MeO2C
MeO2C
N
N
P
OEt
O
O
8
7
F3C
CH3
O
F3C
CH3
O
OEt
OEt
MeO2C
MeO2C
N
H
N
H
P
OEt
O
O
10
9
F3C
Ph
O
F3C
Ph
O
OEt
OEt
MeO2C
MeO2C
N
H
N
H
P
OEt
O
O
12
11
F3C
Bz
O
F3C
Bz
O
OEt
MeO2C
OEt
N
H
MeO2C
N
H
O
P
OEt
O
13
14
- 188 -
List of compounds
F3C
Allyl
O
F3C
Allyl
O
OEt
OEt
MeO2C
MeO2C
N
H
N
H
P
OEt
O
O
16
15
MeO2C
CF3
O
MeO2C
CF3
O
OEt
OEt
N
H
N
H
O
N
P
OEt
O
N
H
H
17
MeO2C
18
CF3
O
MeO2C
CF3
O
OEt
OEt
N
H
N
H
N
O
CH3
N
P
OEt
O
CH3
H
H
20
19
O
O
CF3
CF3
OEt
OEt
N
H
O
O
CO2Me
N
H
CO2Me
O
22
21
- 189 -
P
OEt
O
List of compounds
O
O
CF3
CF3
OEt
OEt
N
H
N
N
H
N
CO2Me
OEt
CO2Me
O
O
Me
Me
24
23
O
O
F3 C
H3C
F3 C
H3C
CO2Me
CO2Me
OEt
OEt
N
H
N
H
N
H
P
H
O
O
N
O
Ph
Ph
26
25
O
O
CF3
CF3
OEt
Me2N
N
H
CO2Me
OEt
Me2N
N
H
H
O
OEt
O
28
O
O
F3C
OEt
P
OEt
MeO2C
P
CO2Me
27
F3C
OEt
N
O
N
P
MeO2C
N
H
OEt
N
O
29
30
- 190 -
List of compounds
O
F3C
F3C
OEt
O
Bz
P
OH
OEt
MeO2C
MeO2C
N
N
H
O
H
32
31
MeO2C
O
CF3
O
OH
P
N
H
P
O
N
OEt
OEt
OEt
OEt
H
O
33
34
O
O
MeO
P
P
OEt
OEt
P
OEt
OEt
H2C
OEt
OEt
P
OEt
OEt
O
O
36
35
O
O
P
P
OEt
P
OEt
OEt
P
OEt
OEt
OEt
OEt
OEt
O
O
38
37
- 191 -
List of compounds
N3
N3
39
F
F
40
F
F
F
F
N3
N3
O
F
F
F
F
F
F
F
O
42
41
O
O
OEt
P
OEt
OEt
P
N
N
N
N
O
OEt
N
O
46
45
O
P
O
OEt
P
OEt
P
N
C6F13
N
N
OEt
P
OEt
N
OEt
P
OEt
O
OEt
P
N
OEt
O
O
N
N
48
47
- 192 -
OEt
OEt
OEt
OEt
O
List of compounds
O
O
NH
P
OEt
OEt
AcO
O
OEt
P
N
AcO
AcO
N
O
OH
P
O
OEt
N
OAc
O
N
O
P
N
N
O
50
O
O
OH
P
OH
P
N
N
C6F13
N
OH
P
N
O
H
51
O
OH
OH
P
N
H
N
OH
OH
N
N
O
53
EtO
EtO
N
O
O
P
P
N
OEt
OEt
N
N
N
N
54
- 193 -
OEt
OEt
N
O
52
P
OEt
OEt
OH
OEt
OEt
N
49
P
OEt
OEt
List of compounds
EtO
EtO
O
O
P
P
OEt
OEt
OAc
OAc
O
O
AcO
AcO
N
N
N
N
OAc
OAc
OAc
N
AcO
N
55
EtO
EtO
N
C6F13
O
O
P
P
N
OEt
OEt
N
N
N
C6F13
N
56
N
N
N
x NaBr
N
N
N
(EtO)2P
O
(EtO)2P
P(OEt)2
O
O
P(OEt)2
O
57
N
N
N
N
N
(EtO)2P
O
x NaBr
N
(EtO)2P
P(OEt)2
O
O
58
- 194 -
P(OEt)2
O
List of compounds
F
F
F
F
N
N
N
(EtO)2P
P(OEt)2
O
N
N
N
(EtO)2P
x NaBr
O
O
P(OEt)2
O
59
O
F
O
F
P
O
OSiMe3
P
F
O
F
P
P
OSiMe3
OSiMe3
OSiMe3
OEt
OEt
OEt
OEt
O
O
61
60
O
F
O
F
P
P
OH
OH
OH
OH
O
62
Compounds 19, 20, 40 and 53 were done in The Russian Academy of Science in
Moscow.
- 195 -
Appendix
Chapter 9
Appendix
9.1
Acknowledgement
9.2
Curriculum Vitae
9.3
Achievements
9.4
List of figures
9.5
List of schemes
9.6
List of tabels
9.7
List of MMPs
- 196 -
Appendix
Acknowledgment
I would like to extend my sincere gratitude and appreciatation to all those who gave
me the possibility to complete this doctor thesis.
First of all, I owe my deepest gratitude to my supervisor Prof. G.-V. Röschenthaler for
his support, benefits and guidance. I feel grateful for introducing me into the world of
phosphorus chemistry.
Secondly, I am highly indebted to Prof. S. N. Osipov from The Russian Academy of
Science in Moscow for his genuine commitment on my work, support, never-failing
enthusiastic attitude towards research.
Special thanks to Prof. I. L. Odinets from The Russian Academy of Science in
Moscow and Prof. E. Breuer from School of Hebrew University in Jerusalem for
cooperation and useful advices.
I would like to acknowledge Mrs. D. Vorob’eva from The Russian Academy of
Science in Moscow who has synthesized compounds 19, 20, 40 and 53.
Thirdly, I would like to thank the members of my research group: Dr. N. Kalinovich, V.
Vogel (Dipl.-Chem.), O. Kazakova (Dipl.-Chem.), K. Vlasov (Dipl.-Chem.), Dr. J.
Ignatowska.
Special thanks are to Mrs. K. Kenst for excellent technical assistance.
Besides
I
would
like
to
thank
to
Dr.
E.
Lork,
Mr.
P.
Brackmann,
and Dr. N. Kalinovich for X-ray analysis, calculations and discussion.
Next, I would like to acknowledge Dr. T. Dülcks and Mrs. D. Kemken for Mass
Spectra analysis.
- 197 -
Appendix
My special gratefulness to my dear husband Filippos, whose patient love, great help,
deep belief and encourangement enabled me to complete this thesis. Besides, I am
very thankful to my parents Bozena & Lucjan Grzeskowiak for all their care and
support.
This work was financially supported by the Deutsche Forschungsgemeinschaft, the
German Israel Foundation of Science and Development and the Russian Foundation
of Basic Research.
- 198 -
Appendix
Curriculum Vitae
Surname
:
Skarpos
Prename
:
Hanna
Maiden name
:
Grzeskowiak
Date of Birth
:
23.10.1979
Place of Birth
:
Rawicz, Polen
Marital status
:
married
Adress
:
Westerreihe 11
27472 Cuxhaven
Deutschland
Cell phone
:
+49-(0)177 / 683 51 24
Email
:
[email protected]
[email protected]
Academic education:
1998 - 2003
Faculty of Chemistry, Adam Mickiewicz University,
Poznan, Poland
Master Thesis: „Preparation of Halogene Derivatives of
Guanosine and Uracil Using Vilsmeier Procedure and
Wittig Reaction“ - under supervision of Prof. H. Koroniak
2002 - 2003
Pedagogical course, specialization chemistry, Faculty of
Chemistry, Adam Mickiewicz University, Poznan, Poland
2002
Socrates-Erasmus Exchange Program, Department of
Chemistry, Aristotle University of Thessaloniki, Greece
2000 - 2004
The Poznan College of Modern Languages, Poznan,
Poland, English philology
2004 - 2007
Institut of Inorganic and Physical Chemistry, Bremen
University, doctoral studies
- 199 -
Appendix
Professional qualification:
Languages
Polish (mother tongue), English and Russian fluent in
speaking and writing, German, studying French and Greek
Computer literacy
MS Word, MS Excel, MS Power Point, ChemDraw, ISIS
Draw, Beilstein, SciFinder, MestReC; work with MS
Windows XP and Vista as well as Mac OS X
Publications:
“An Easy Transformation of 2-Amino-2- (hydroxyimino) acetates to
Carbamoylformamidoximes”
D. N. Nicolaides, K. E. Litinas, T. Papamehael, H. Grzeskowiak, D. R. Gautam, K. C.
Fylaktakidou
Synthesis 2005, 407-410
“Methyltrifluoropyruvate imines possesing N-oxalyl and N-phosphonoformyl groupsprecursors to a variety of Į- CF3- Į- amino acids derivatives”
H. Skarpos, D. V. Vorob’eva, S. N. Osipov, I. L. Odinets E. Breuer, G.- V.
Röschenthaler
Organic and Biomolecular Chemistry, 2006, 4, 3669- 3674
„Synthesis of functionalized N- Bisphosphonates via click chemistry“
H. Skarpos, S. N. Osipov, D. V. Vorob’eva, I. L. Odinets E. Lork, G. - V.
Röschenthaler
Organic and Biomolecular Chemistry, 2007, 5, 2361- 2367
- 200 -
Appendix
Achievements
PUBLICATIONS
“An Easy Transformation of 2-Amino-2- (hydroxyimino) acetates to
Carbamoylformamidoximes”
D. N. Nicolaides, K. E. Litinas, T. Papamehael, H. Grzeskowiak, D. R. Gautam, K. C.
Fylaktakidou
Synthesis 2005, 407-410
“Methyltrifluoropyruvate imines possesing N-oxalyl and N-phosphonoformyl groups precursors to a variety of Į- CF3- Į- amino acids derivatives”
H. Skarpos, D. V. Vorob’eva, S. N. Osipov, I. L. Odinets, E. Breuer, G.-V.
Röschenthaler
Organic and Biomolecular Chemistry, 2006, 4, 3669- 3674
“Synthesis of functionalized bisphosphonates via click chemistry”
H. Skarpos, S. N. Osipov, D.V. Vorob’eva, I.L. Odinets, E. Lorkr, G.-V. Röschenthaler
Organic and Biomolecular Chemistry, 2007, 5, 2361-2367
POSTER PRESENTATIONS
ƒ 18th International Symposium on Fluorine Chemistry
Bremen, Germany, July 30 – August 4, 2006
H. Skarpos, D. V. Vorob’eva, S. N. Osipov, I. L. Odinets E. Breuer, G. -V.
Röschenthaler
“Methyltrifluoropyruvate imines possesing N-oxalyl and N- phosphonoformyl groupsprecursors to a variety of Į- CF3- Į- amino acids derivatives”
ƒ 15th European Symposium on Fluorine Chemistry
Prag, Czech Republic, July 15 - 20, 2007
H. Skarpos, G.-V. Röschenthaler
“Synthetic route to novel fluorinated bisphosphonates”
- 201 -
Appendix
ORAL PRESENTATIONS
ƒ 12. Deutsche Fluortag
Schmitten, Germany, September 4-6, 2006
H. Skarpos, D. V. Vorob’eva, S. N. Osipov, I. L. Odinets E. Breuer, G. -V.
Röschenthaler
“A simple pathway to N-oxalyl and N-phosphonoformyl derivatives of Į-Tfm-Į-amino
acids”
ƒ 10th. Northern-German Doctoral Student Colloquium of Inorganic Chemistry
Bremen, Germany, September, 26-28, 2007
H. Skarpos, S. N. Osipov, G.-V. Röschenthaler
„A new class of nitrogen-containing bisphosphonates (N-BPs). Application of the click
chemistry”
ƒ Winter Kolloquium GDCh Junger Chemiker
Bremen, Germany, December, 17, 2007
H. Skarpos, S. N. Osipov, G.-V. Röschenthaler
„A new class of nitrogen-containing bisphosphonates (N-BPs). Application of the click
chemistry”
BESIDES RESULTS OF THIS WORK WERE PRESENTED AT:
ƒ 18th Winter Fluorine Conference
St. Petersburg, FL (USA), January 14-19, 2007
H. Skarpos, D. Vorob’eva, S. Osipov, I. Odinets, E. Breuer, G.-V. Röschenthaler,
“Methyl trifluoropyruvate imines possessing N-oxalyl or N-phosphonoformyl groups”
ƒ 15th European Symposium on Fluorine Chemistry
Prag, Czech Republic, July 15- 20, 2007
H. Skarpos, N. E. Shevchenk, D. Vorob’eva, V. G. Nenajdenko, S. Osipov, I. Odinets,
G.- V. Röschenthaler
“Trifluoropyruvate. A versatile reactant in organic chemistry”
- 202 -
Appendix
List of figures
Figure 1
The effect of fluorine substitution on drug discovery
Figure 2
Structural domains of MMPs
Figure 3
Hydroxamate Inbibitors of MMPs: Barimastat and Marimastat
Figure 4
Bisphosphonate Inhibitors of MMPs: Alendronate and Tiludronate
Figure 5
ZBGs (Zinc- Binding groups) commonly used in designing of MMPIs
Figure 6
Counterparts for Cu-catalyzed azide-alkyne 1,3-dipolar cycloaddition
Figure 7
Structure of amino acids (AAs)
Figure 8
Ethyl oxamate and diethylcarbamoylphosphonate - active ZBGs
Figure 9
Coordination of the carbamoylphosphonate to a metal ion: (I) monodentate
manner, (II) four- membered ring, (III) five-membered ring between the
oxygen’s of the two functional group
Figure 10
Main classes of Tfm-AAs; A: Trifluoromethylated Į-amino acids, B:
Trifluoromethylated ȕ-amino acids, C: Trifluoromethylated cyclic amino acids
Figure 11
Methyl trifluoropyruvate imines possessing sulfonyl and phosphoryl groups
Figure 12
Orbitals structures of methyltrilfuoropyruvate imines 7 and 8
Figure 13
X-ray structure of the compound 17 methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2(1H-indol-3-yl)alaninate
Figure 14
Another view of the compound 17
Figure 15
X-ray structure of the compound 21 methyl N-[ethoxyoxalyl]-3,3,3-trifluoro-2(2-furyl)alaninate, showing the atom-numbering scheme.
Figure 16
The molecular structure of 21 showing the two hydrogen bonding interactions
Figure 17
Spectra: 19F NMR (CDCl3) and 31P NMR (CDCl3) of the compound 30 methyl
3-(diethylphosphonyl)-2-(trifluoromethyl)-2H-azirine-2-carboxylate
Figure 18
Synthetic protocols for 2H-azirines
Figure 19
Activated 3-trifluoromethyl-aziridine-2-hydroxamates- inhibitor of MMPs
Figure 20
Bisphosphonates as structural analogues of diphosphates
Figure 21
Structure-activity relationships in geminal bisphosphonates
Figure 22
Mechanism of bisphosphonates to bind to divalent metal ions such as Ca2+
- 203 -
Appendix
Figure 23
Non-nitrogen-BPs currently used in clinical setting
Figure 24
Nitrogen-BPs currently used in clinical setting
Figure 25
TLC presenting Rf values for compounds 37 and 38
Figure 26
X-ray structure of the compound 38, showing the atom-numbering scheme
Figure 27
Resonance structures of azides R-N3
Figure 28
The set of ligands used in the synthesis of aryl azides
Figure 29
Most common copper system used in click chemistry
Figure 30
Topological and electronic similarities of amides and 1,2,3-triazoles
Figure 31
X-ray structure of the compound 54
Figure 32
F3-Etidronic acids - fluorinated analogue of Etidronate
Figure 33
19
Figure 34
31
Figure 35
19
Figure 36
31
Figure 37
3,3,3-trifluoroalanine derivatives bearing on the nitrogen atom N-oxalyl and Nphosphonoformyl potential ZBGs groups
Figure 38
Aza-analogue of Zolendronic acid
Figure 39
Difluoromethylidene derivative containing phosphonooxy and phosphonate
groups
NMR spectra of the compound 61, signals due to two fluor nuclei
NMR spectra of the compound 61, signals due to two phosphorus nuclei
NMR spectra of the compound 62, signals due to two fluor nuclei
NMR spectra of the compound 62, signals due to two phosphorus nuclei
- 204 -
Appendix
List of schemes
Scheme 1
Design of N-oxalyl and N-phosphonoformyl derivatives of Į-Tfm-Į-amino
acids - potential inhibitors of MMPs
Scheme 2
A general synthetic approach to R-functionalized nitrogen containing
bisphosphonates (N-BPs)
Scheme 3
A general synthetic approach to R-functionalized nitrogen-bisphosphonates
(N-BPs) containing two triazoles moieties
Scheme 4
A general synthetic approach to nitrogen-bisphosphonate (N-BPs) based on
one pot reaction
Scheme 5
A general synthetic approach to fluorinated bisphosphonates (FBPs) based on
Arbuzov/ Perkov reaction
Scheme 6
Synthesis of an active ZBG-diethyl carbamoylphosphonate 4 via Arbuzov
reaction
Scheme 7
Synthesis of methyl 2,3,3,3-tetrafluoro-2-methoxypropanoate by methanolysis
of 1,1,2,3,3,3- hexafluoropropanoate (HFPO) (A); Synthesis of methyl 3,3,3trifluorpyruvate from methyl 2,3,3,3-tetrafluoro-2-methoxypropanoate (B)
Scheme 8
Synthetic route to methyltrilfuoropyruvate imine 7 bearing on nitrogen potential
ZBG, namely oxalyl group
Scheme 9
Synthetic route to methyltrilfuoropyruvate imine 8 bearing on nitrogen potential
ZBG, namely phosphonoformyl group
Scheme 10
Reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines
with different Grignard reagents
Scheme 11
Reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines
with indoles
Scheme 12
Reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines
with furan and N-methylpyrrole
Scheme 13
Reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines
with 1-phenyl-3-methylpyrazol-5-on
Scheme 14
Reactions of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines
with N,N-dimethylaniline
Scheme 15
Reduction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate imines
with NaBH4
Scheme 16
Proposed mechanism of the formation of azirine cycle for the compound 30
methyl 3-(diethylphosphonyl)-2-(trifluoromethyl)-2H-azirine-2-carboxylate
- 205 -
Appendix
Scheme 17
Transformation of the imine 8 into azirine 30 methyl 3-(diethylphosphonyl)-2(trifluoromethyl)-2H-azirine-2-carboxylate
Scheme 18
Hydrolysis of the oxamate moiety into oxamic acid 32
Scheme 19
Hydrolysis of the oxamate moiety into oxamic acid 33
Scheme 20
Products of thermal 1,3-cycloaddition
Scheme 21
Copper(I)-catalyzed synthesis of 1,4- disubstitued 1,2,3-triazoles
Scheme 22
Synthesis of propargyl-substitued bisphosphonates
Scheme 23
Synthesis of tetraethyl ethylidenebis(phosphonate) 36
Scheme 24
A convenient synthetic way to bispropargyl bisphosphonate 38
Scheme 25
Cyclopolymerization of 38 by methathesis polymerization technique
Scheme 26
Synthesis of aryl azides
Scheme 27
Synthesis of fluorinated azide
Scheme 28
1,3-dipolar cycloaddition between monopropargyl bisphosphonates and
different azides
Scheme 29
Generation of Cu(I) in situ from CuSO4x5H2O
Scheme 30
Proposed catalytic cycle for the Cu(I) catalyzed ligation
Scheme 31
1,3-Cycloaddition of dipropargyl bisphosphonate 38 with different azides
yielding bistriazoles
Scheme 32
Synthesis
of
tetraethyl
2-(1-benzyl-1H-1,2,3-triazol-4-yl)ethane-1,1diyldiphosphonate 57 via one pot reaction
Scheme 33
Synthesis of tetraphosphonates molecules via one pot reaction
Scheme 34
Hydrolysis of the ester groups at the phosphorus atom
Scheme 35
Selective removing of pivaloylmethyl group from 52 followed by hydrolysis of
the ester groups at the phosphorus atom from 53
Scheme 36
Synthesis of 1-hydroxy-2,2,2-trifluoroethylidene-bisphosphonic acid
Scheme 37
The Michealis-Arbuzov reaction201, 202
Scheme 38
The Perkow reaction203
Scheme 39
Synthesis of F-1-alkene-1-phosphonate
Scheme 40
Synthetic route to difluoro(phosphonooxy)vinylphosphonate 61 and 62
Scheme 41
Reaction mechanism (Perkow/Arbuzov)
- 206 -
Appendix
Scheme 42
Conversion of the ester into free acid 63
Scheme 43
General synthetic route to methyl 2-oxalylimino and 2-(phosphonformimido)3,3,3-trifluoropropanoates
Scheme 44
General synthetic route Į-Tfm-Į-amino acids derivatives
Scheme 45
A new class of nitrogen containing bisphosphonates (N-BPs)
Scheme 46
Synthetic pathway to monopropargyl bisphosphonate
Scheme 47
N-Bisphosphonates molecules with ditriazoles moieties
Scheme 48
N-Bisphosphonates molecules with ditriazoles moieties via one pot reaction
- 207 -
Appendix
List of tables
Table 1
Results
of
the
reaction
of
N-oxalyl
and
N-phosphonoformyl
methyltrifluoropyruvate imines with Grignard reagents via Scheme 10
Table 2
Results of reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate
imines with indols via Scheme 11
Table 3
Results of reaction of N-oxalyl and N-phosphonoformyl methyltrifluoropyruvate
imines with furan and N-methylpyrrol via Scheme 12
Table 4
Results
of
reaction
between
N-oxalyl
and
N-phosphonoformyl
methyltrifluoropyruvate imines and 1-phenyl-3-methylpyrazol-5-on and N,Ndimethylaniline via Scheme 13 and 14
Table 7
Selected geometric parameters for the compound 17
Table 6
Hydrogen bonds for 21 [pm and o]
Table 7
Selected geometric parameters for the compounds 21
Table 8
Selected geometric parameters for the compound 38
Table 9
Reaction of monopropargyl bisphosphonate with different azide
Table 10
Selected geometric parameters for the compound 54
- 208 -
Appendix
List of matrix metalloproteinases (MMPs)
MMPs ʋ
Matrix substrates or functions
Interstistial collagenases
( EC 3.4-24.7)
MMP-1
Collagens I, II, II, VII, X, gelatins, entactin,
aggrecan, link protein
Neutrophil collagenase
(EC 3.4-24.34)
MMP-8
Collagens I, II, II, aggrecan, link protein
Collagenase 3
MMP-13
MMP-13
Collagens I, II, III
Collagenase 4
(Xenopus)
MMP-18
Collagen I
Gelatinase A
(EC 4.3-24.24)
MMP-2
Gelatins, collagens I, IV, V, VII, X, XI, fibronectin,
laminin, aggrecan, elastin, large tenascin C,
vitronectin, ȕ-amyloid protein precursor (ȕsecretase-like activity)
Gelatinase B
(EC 3.4-24-35)
MMP-9
Gelatins, collagens IV, V, XIV, aggrecan, elastin,
entacin, vitronectin
MMP-3
Aggrecan, gelatins, fibronectin, laminin, collagen III,
IV, IX, X, large tenascin, vitronectin
MMP-10
Aggrecan, fibronectin, collagen IV
Enzyme
Collagenases
Gelatinases
Stromelysins
Stromelysin 1
(EC 3.4-24.17)
Stromelysin 2
(EC 3.4-24.22)
Membrane-type MMPs
- 209 -
Appendix
Collagens I, II, III, fibronectin, laminin-1, vitronectin,
dermatan sulfate proteoglycan: active proMMP-2
and proMMP-13
MT1-MMP
MMP-14
MT2-MMP
MMP-15
MT3-MMP
MMP-16
Actives proMMP-2
MT4-MMP
MMP-17
Not known
Matrilysin
(EC 3.2.24.23)
MMP-7
Aggrecan, fibronectin, laminin, gelatins, collagen IV,
elastin, entacin, small tenascin-C, vitronectin
Stromelysin 3
MMP-11
Weak acitivity on fibronectin, laminin, collagen IV,
aggrecan, gelatins
Metalloelastase
MMP-12
Elastin
RASI
MMP-19
Aggrecan, COMP
Enamelysin
MMP-20
Amelogenin, Aggrecan, COMP
no name
MMP-21
Alpha-1 Antitrypsin
Femalysin
MMP-23
Not identified so far. Expressed in ovary, testis
MT5-MMP
MMP-24
Activates Progelatinase A, degrades Proteoglycans
MT6-MMP
MMP-25
Activates Progelatinase A
Matrilysin 2
MMP-26
Activates Progelatinase B, degrades other ECM
components
Epilysin
MMP-28
Casein
Not known
Others
- 210 -
Appendix
- 211 -